In vitro models of the blood-brain barrier using iPSC-derived cells · 2019-11-19 · In vitro...

In vitro models of the blood-brain barrier using iPSC-derived

cells

Louise Delsing

Department of Psychiatry and Neurochemistry

Institute of Neuroscience and Physiology

Sahlgrenska Academy, University of Gothenburg

Gothenburg 2019

Delsing, Louise

Cover illustration: Human induced pluripotent stem cell-derived brain

endothelial cells stained with a green fluorescent antibody for the glucose

transporter Glut-1.

In vitro models of the blood-brain barrier using iPSC-derived cells

© Louise Delsing 2019

[email protected]

ISBN: 978-91-7833-634-0 (PRINT)

ISBN: 978-91-7833-635-7 (PDF)

http://hdl.handle.net/2077/61824

Printed in Gothenburg, Sweden 2019

Printed by BrandFactory

To Daniel

Delsing, Louise

“In nature, nothing exists alone”

- Rachel Carson

i


Louise Delsing

Department of Psychiatry and Neurochemistry, Institute of Neuroscience and

Physiology, Sahlgrenska Academy, University of Gothenburg

Gothenburg, Sweden

ABSTRACT

The blood-brain barrier (BBB) constitutes the interface between the blood and the

brain tissue. Its primary function is to maintain the tightly controlled

microenvironment of the brain. Models of the BBB are useful for studying the

development and maintenance of the BBB as well as diseases affecting it. Furthermore,

BBB models are important tools in drug development and support the evaluation of

the brain-penetrating properties of novel drug molecules. Currently used in vitro

models of the BBB include immortalized brain endothelial cell lines and primary brain

endothelial cells of human and animal origin. Unfortunately, these cell lines and

primary cells have failed to recreate physiologically relevant control of transport in

vitro. Human-induced pluripotent stem cell (iPSC)-derived brain endothelial cells have

proven a promising alternative source of brain endothelial-like cells that replicate tight

cell layers with low para-cellular permeability. Given the possibility to generate large

amounts of iPSC-derived brain endothelial cells they are a feasible alternative when

modelling the BBB in vitro.

This thesis aimed to develop iPSC-derived models of the BBB that display a barrier

like phenotype and characterize these models in terms of specific properties. The BBB

model development was based on investigations into mechanisms important for barrier

formation in iPSC-derived endothelial cells and development of high-quality

supporting cells. The possibilities to use the model in drug discovery, and in

determination of brain penetrating capacity of drug substances were specifically

considered. These studies have increased knowledge of molecular mechanisms behind

the restricted permeability across iPSC-derived endothelial cells and identified

transcriptional changes that occur in iPSC-derived endothelial cells upon coculture

with relevant cell types of the neurovascular unit. Furthermore, high quality iPSC-

Delsing, Louise

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derived astrocytic cells were developed, and the biological relevance and model

diversity between astrocytic models were evaluated. Both astrocytes and brain

endothelial cells have been adapted to xeno-free culture conditions and used in the

BBB models, demonstrating a xeno-free BBB model. Finally, a more biologically

relevant microphysiological dynamic BBB model was generated. This model

demonstrated improved permeability modelling and compatibility with high-

throughput substance permeability screening.

Taken together these results show that iPSC-derived BBB models are useful for

studying BBB-specific properties in vitro and that both marker expression and

functional evaluation of iPSC-derived cells are important in assessing cell identity and

cell quality. In addition, these results show that iPSC derived BBB models are feasible

for high-throughput permeability studies.

Keywords: Blood-brain barrier, iPSC, in vitro model, permeability

ISBN: 978-91-7833-634-0 (PRINT)

ISBN: 978-91-7833-635-7 (PDF)

iii

SAMMANFATTNING PÅ SVENSKA

Blod-hjärnbarriärens (BHB) huvudsakliga uppgift är att skydda det känsliga centrala

nervsystemet från potentiellt skadliga substanser som cirkulerar i blodet. Genom att

begränsa permeabiliteten av blodkärlen i hjärnan bibehålls den specifika miljön i

centrala nervsystemet som krävs för att hjärnan ska fungera optimalt. Eftersom BHB

är en vital del av det centrala nervsystemet är det svårt att studera BHB direkt i

människokroppen utan att göra skada. Därför behövs modeller.

Modeller av BHB är viktiga för att studera utvecklingen och upprätthållandet av BHB

och även för att förutsäga i vilken utsträckning nya medicinska molekyler kommer att

ta sig in i centrala nervsystemet. Ofta används cellbaserade BHB-modeller uppbyggda

av hjärnendotelceller med eller utan pericyter och nervceller. Immortaliserade cellinjer

av humana hjärnendotelceller och primära hjärnendotelceller från djur har använts.

Tyvärr uppvisar dessa cellmodeller inte en tät barriär likt den i människa när de odlas

i laboratoriemiljö (in vitro). Det finns även bevis för att BHB skiljer sig åt mellan

människa och djur, vilket medför att modeller som baseras på djurceller kan vara

missvisande. Därtill pågår stora ansträngningar för att reducera djurförsök inom

forskningen. Sammanfattningsvis behövs det nya och bättre in vitro-modeller för att

ingående kunna studera egenskaper hos den mänskliga BHB i laboratorier.

Mänskliga inducerade pluripotenta stamceller (iPSC) skapas genom att celler från en

vuxen person återprogrammeras till ett tidigt utvecklingsstadium där dessa kan bilda

alla olika celltyper i kroppen. Hjärnendotelceller som bildats från iPSC har visat sig

återskapa en mycket tät barriär i laboratoriemiljö. En av de mest typiska egenskaperna

för iPSC är att de har hög delningsfrekvens. Genom att använda iPSC kan stora

mängder mänskliga hjärnendotelceller med barriäregenskaper lika de i BHB

produceras och användas för att studera specifika egenskaper hos den mänskliga BHB.

Syftet med denna avhandling var att utveckla BHB-modeller från iPSC och undersöka

BHB-specifika egenskaper hos dessa modeller. Modellerna har utvärderats med

avseende på faktorer som påverkar barriäregenskaper hos hjärnendotelceller. Särskild

hänsyn har tagits till modellernas förmåga att användas i läkemedelsutveckling för att

studera hjärnexponeringen av nya medicinska molekyler. Specifik identitet för olika

celltyper har utvärderats genom att undersöka uttryck av gener och protein som

kännetecknar dessa celltyper. Funktionalitet hos cellerna har studerats genom att

undersöka deras förmåga att utföra processer som normalt utförs av dessa celler i

kroppen. Både passiv och aktiv permeabilitet över BHB-modellen studerades med

hjälp av fluorescerande verktygssubstanser och kända läkemedelssubstanser.

Delsing, Louise

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När hjärnendotelceller från iPSC odlas tillsammans med pericyter och nervceller

reducerades permeabilitet i BHB-modeller. Avhandlingens resultat har ökat

förståelsen för vilka molekylära mekanismer som bidrar till denna reducerade

permeabilitet. Högkvalitativa astrocyter har skapats från iPSC och jämförts med andra

astrocytmodeller för att utvärdera deras relevans som human astrocytmodell samt för

att förstå skillnader mellan olika, vanligt förekommande, astrocytmodeller. Produktion

av både astrocyter och hjärnendotelceller från iPSC har anpassats till

odlingsbetingelser utan användning av animaliska biprodukter. Astrocyter och

hjärnendotel celler har sedan använts i BHB-modeller för att skapa en helt human

modell. Slutligen har BHB-modellen förbättrats ytterligare genom utveckling av en

mer biologiskt relevant modell som återskapar den tredimensionella miljön som råder

i hjärnans blodkärl. I denna modell växer hjärnendotelceller i ett artificiellt kärl där de

fysiska påfrestningarna av blodflöde simuleras med hjälp av genomströmning av

endotelcellskärlet. Modelleringen av specifik transport som framför allt påverkar

läkemedelssubstanser förbättrades i denna modell. Denna modell lämpar sig även för

storskalig permeabilitetsanalys.

Sammantaget visar resultaten i denna avhandling att BHB-modeller som är uppbyggda

av celler från iPSC är mycket användbara för att studera BHB-specifika egenskaper i

laboratoriemiljö. Dessutom tydliggörs hur analyser av både proteinuttryck och

funktionalitet är viktigt för att utvärdera kvaliteten hos specifika celltyper, samt för

analysen av deras förmåga att utföra sina respektive uppgifter. Därtill visas att

storskalig analys av BHB-permeabilitet är möjlig med BHB-modeller från iPSC.

v

LIST OF PAPERS

This thesis is based on the following studies, referred to in the text by their Roman

numerals.

I. Delsing L, Dönnes P, Sánchez J, Clausen M, Voulgaris D, Falk A,

Herland A, Brolén G, Zetterberg H, Hicks R, and Synnergren J. Barrier

Properties and Transcriptome Expression in Human iPSC-Derived

Models of the

Blood-Brain Barrier.

Stem Cells. 2018 Dec;36(12):1816-1827.

II. Lundin A, Delsing L, Clausen M, Ricchiuto P, Sanchez J, Sabirsh A,

Ding M, Synnergren J, Zetterberg H, Brolén G, Hicks R, Herland A and

Falk A. Human iPS-Derived Astroglia from a Stable Neural Precursor

State Show Improved Functionality Compared with Conventional

Astrocytic Models.

Stem Cell Reports. 2018 Mar;10(3):1030-1045.

III. Delsing L, Kallur T, Zetterberg H, Hicks R, and Synnergren J. Enhanced

Xeno-Free Differentiation of hiPSC-Derived Astroglia Applied in a

Blood-Brain Barrier Model

Fluids and Barriers of the CNS. 2019 Aug;16(1):27.

IV. Delsing L, Zetterberg H, Herland A, Hicks R and Synnergren J. A

Human iPSC-Derived Microphysiological Blood-Brain Barrier Model

for Permeability Screening

Manuscript

Delsing, Louise

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vii

Contents SAMMANFATTNING PÅ SVENSKA .................................................................... III

LIST OF PAPERS ........................................................................................... V

ABBREVIATIONS ............................................................................................. IX

1 THE BIOLOGY OF THE BLOOD-BRAIN BARRIER ........................................... 1

Early brain development ....................................................................... 2

Blood-brain barrier development .......................................................... 3

Brain endothelial cells ........................................................................... 4

Astrocytes .............................................................................................. 5

Pericytes ................................................................................................ 7

Neurons and microglia .......................................................................... 8

The basement membrane ....................................................................... 9

The blood-brain barrier and disease .................................................... 10

The blood-brain barrier in drug discovery .......................................... 11

2 PERMEABILITY OF THE BLOOD-BRAIN BARRIER ....................................... 13

Inter-cellular junctions ........................................................................ 14

Transport proteins ............................................................................... 15

Efflux transporters ............................................................................... 16

Modulating blood-brain barrier transport for therapeutic purposes .... 17

3 IPSC-DERIVED CELLS FOR BLOOD-BRAIN BARRIER MODELING ............... 19

iPSC-derived endothelial cells ............................................................ 20

iPSC-derived pericytes ........................................................................ 21

iPSC-derived astrocytes ...................................................................... 22

4 BLOOD-BRAIN BARRIER MODELS ............................................................. 25

Characterization of in vitro BBB models ............................................ 27

iPSC-derived blood-brain barrier models ........................................... 29

Microfluidic models ............................................................................ 31

5 AIMS ......................................................................................................... 35

6 METHODOLOGICAL CONSIDERATIONS ..................................................... 37

Ethics ................................................................................................... 37

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Cells .................................................................................................... 37

Differentiation of iPSC-derived endothelial cells ............................... 38

Differentiation and functional characterization of iPSC-derived

astrocytes .................................................................................................... 38

Characterization of protein and mRNA expression ............................ 39

Barrier integrity assays ........................................................................ 40

RNA sequencing and pathway analysis .............................................. 41

Microphysiological culture systems .................................................... 41

Statistical analysis ............................................................................... 42

7 SUMMARY OF FINDINGS ........................................................................... 45

Paper I: Barrier Properties and Transcriptome Expression in Human

iPSC-Derived Models of the Blood-Brain Barrier...................................... 45

Paper II: Human iPS-Derived Astroglia from a Stable Neural Precursor

State Show Improved Functionality Compared with Conventional Astrocytic

Models ........................................................................................................ 46

Paper III: Enhanced Xeno-Free Differentiation of hiPSC-Derived

Astrocytes Applied in a Blood-Brain Barrier Model .................................. 48

Paper IV: An iPSC-Derived Microphysiological Blood-Brain Barrier

Model for Permeability Screening .............................................................. 49

8 DISCUSSION .............................................................................................. 53

iPSC-derived blood-brain barrier models ........................................... 53

Comparison to other iPSC-derived blood-brain barrier models.......... 54

Efflux assessment in iPSC-derived blood-brain barrier models ......... 56

Blood-brain barrier phenotype of iPSC-derived endothelial cells ...... 57

Brain permeability prediction in drug discovery ................................ 58

Limitations .......................................................................................... 60

9 CONCLUSIONS .......................................................................................... 63

10 FUTURE PERSPECTIVES ............................................................................. 65

ACKNOWLEDGEMENT .................................................................................... 69

REFERENCES .................................................................................................. 71

ix

ABBREVIATIONS

2D Two Dimensional

3D Three Dimensional

Aβ Amyloid Beta

ABC ATP-Binding Cassette

AD Alzheimer’s Disease

AJ Adherens Junction

ALDH1L1 Aldehyde Dehydrogenase 1 Family Member L1

Ang-1 Angiopoietin 1

ANOVA Analysis of Variance

apoE Apolipoprotein E

AQP4 Aquaporin 4

BBB Blood-Brain Barrier

BCRP Breast Cancer Resistance Protein

BLBP Brain Lipid-Binding Protein

BM Basement Membrane

BMP Bone Morphogenetic Protein

Caco-2 Human Epithelial Colorectal Adenocarcinoma Cells

CCF CCF-STTG1 Astrocytoma Cell Line

CD31 Cluster of Differentiation 31

CMT Carrier Mediated Transport

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CNS Central Nervous System

CSF Cerebrospinal Fluid

EAAT Excitatory Amino Acid Transporter

ECM Extra Cellular Matrix

EGF Epidermal Growth Factor

ELISA Enzyme-linked Immunosorbent Assay

ESC Embryonic Stem Cell

FDA Food and Drug Administration

FGF Fibroblast Growth Factor

FPKM Fragments Per Kilobase Million

GDNF Glia Derived Neurotrophic Factor

GFAP Glial Fibrillary Acid Protein

GLN Glutamine

GLU Glutamate

GLUL Glutamine synthetase

GW Gestation Week

HEK Human Embryonic Kidney Cell Line

HIV Human Immunodeficiency Virus

ICC Immunocytochemistry

iCellAstro Commercially Available iPSC-Derived Astrocytes

IGF1 Insulin Like Growth Factor

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IgG Immunoglobulin G

IL6 Interleukin 6

iPSC Induced Pluripotent Stem Cell

kDa Kilo Dalton

L2020 ECM From Murine Sarcoma Cells

LAT Amino Acid Transporter

LN521 Recombinant Laminin 521

LRP-1 Lipoprotein Receptor-Related Protein 1

Lt-NES Long-Term Neuroepithelial Stem Cells

LXR Liver X Receptor

MDCK Madin-Darby Canine Kidney Cell Line

MS Multiple Sclerosis

MPS Microphysiological System

MRP Multidrug Resistance Protein

NES-astro Astrocytes Derived from Lt-NES

NG2 Neuron Glia Antigen 2

NVU Neurovascular Unit

Papp Apparent Permeability

PCA Principal Component Analysis

PDGFRβ Platelet-derived Growth Factor Receptor Beta

P-gp P-Glycoprotein

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phaAstro Primary Human Astrocytes

RA Retinoic Acid

RNAseq RNA Sequencing

RMT Receptor-Mediated Transcytosis

SHH Sonic Hedgehog

SLC Solute Carrier

TEER Trans Endothelial Electrical Resistance

TGF-β Transforming Growth Factor Beta

TJ Tight Junction

TNFα Tumour Necrosis Factor Alpha

VEGF Vascular Endothelial Growth Factor

ZO Zonula Occludens


1

1 THE BIOLOGY OF THE BLOOD-BRAIN BARRIER

The blood-brain barrier (BBB) is the interface between the blood and the brain tissue.

Its primary function is to maintain the tightly controlled microenvironment of the

brain. The barrier consists of endothelial cells with properties specific to the central

nervous system (CNS) (1). These brain endothelial cells control the permeability of

the barrier. At the brain side of the endothelial cells, the extracellular basement

membrane (BM) surrounds the endothelial cells and embeds the pericytes. Astrocytic

end-feet are in contact with the basal membrane. This unit of astrocytes, pericytes,

basal membrane and endothelial cells is often referred to as the neurovascular unit

(NVU, Figure 1) (1). Together these components make up the BBB and govern its

development, maintenance and function. The paracellular tightness of the endothelial

cells in the BBB acts as a physical barrier for cells, proteins and water-soluble agents.

Transporter proteins control nutrient supply and permeability of small molecules in a

specific manner. The BBB is a highly dynamic structure, which is regulated by the

interactions of the cellular and extra cellular matrix (ECM) parts of the NVU. Isolated

primary brain endothelial cells rapidly lose their BBB properties when cultured in vitro

(2), consequently it is plausible that the BBB properties are not intrinsic to the brain

endothelial cells but rather depend on the specific microenvironment that all

components of the NVU create together.

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Figure 1. The neurovascular unit. Endothelial cells are linked together via tight

junctions. On the brain side of the endothelial cell layer the basement membrane

surrounds the endothelial cells and embeds the pericytes. Astrocytic end-feet are in

contact with the endothelial cells.

Early brain development

The human brain is an immensely complex structure that consists of more than 100

billion neurons (3). To support these neurons, different types of glia cells are present,

mainly astrocytes, oligodendrocytes and microglia. While the functions of

oligodendrocytes and microglia are well characterized as myelinating and immune

surveillance respectively, the functions of astrocytes are more diverse, and the list of

tasks performed by astrocytes are growing continuously. For example, astrocytes

maintain brain homeostasis, support accurate synaptic signalling, govern synaptic

formation and promote BBB formation and maintenance (4).

Human brain development is a lengthy process that begins in the third gestation week

(GW) when gastrulation occurs (3). In the gastrulation phase, the three germ layers;


3

ectoderm, mesoderm, and endoderm, are formed. The ectodermal cells give rise to the

CNS, the mesodermal cells give rise to muscle cells, blood cells and the blood vessels

that make up the BBB, and the endoderm give rise to many of the cell types that make

up internal organs such as lung cells and gastrointestinal tracts. The neuroectoderm

develops through three distinct phases, the development of the neural plate, formation

of the neural groove, and finally, folding into the neural tube, which buds of from the

ectodermal tissue (3). The neural tube is regionally specified, the rostral part will give

rise to the brain and the caudal part will give rise to the hind-brain and spinal tube, in

addition the hollow middle part of the neural tube will give rise to the ventricles and is

thus referred to as the ventricular zone. Already at this stage, the vascularization of the

neuroectoderm and the development of the BBB are initiated.

Blood-brain barrier development

The development of the BBB begins when vessels start to invade the developing

neuroectoderm (1). Neural progenitors secrete the strongly angiogenic factor vascular

endothelial growth factor (VEGF), which guides sprouting of vessels into the neural

tissue (5). Neural progenitors also secrete WNT, which is necessary for endothelial

cell migration and induces expression of BBB associated genes, such as the glucose

transporter Glut-1 and tight junction proteins in the endothelial cells (6). Downstream

signalling from WNT is essential for vascularization of the CNS but not peripheral

tissues (6), suggesting a specific role of WNT signalling in development of the brain

vasculature. In addition, WNT-mediated signalling deficits have been identified as a

cause of BBB disruption in iPSC-derived endothelial cells from Huntington’s disease

patients, further emphasizing its importance in BBB development (7). Permeability

restriction occurs already early in development and rodent studies show that the early

embryonic BBB prevents leakage of proteins from the blood to the brain (8). Similar

restriction of blood to brain permeability was recently confirmed in human early

embryos. The first vessels penetrating into the brain parenchyma in the human

embryo restricts permeability of blood-derived molecules and are immunopositive

for claudin-5, suggesting that even the earliest brain blood vessels at GW five have

BBB characteristics (9). Cues from astrocytes and pericytes are essential in BBB

development, and lack of such signals are linked to severe abnormalities of the BBB

(10, 11). Sonic hedgehog (SHH)-signalling is important for BBB formation and SHH

knockout mice display embryonic lethality (10). The vascularization of their CNS is

complete but expression of tight junction (TJ) proteins is reduced, suggesting that SHH

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is important for BBB maturation and tightening. This effect is proposed to be astrocyte-

mediated as SHH is produced and released by astrocytes. Furthermore, SHH-signalling

is important in both embryonic BBB development and adult BBB immunocompetence

(10). Most of the mature astrocytes develop after birth (4). Consequently, astrocyte-

dependent changes to BBB function is likely to continue after birth.

Brain endothelial cells

While only making up 2% of the total body mass, the brain consumes about 20% of

the glucose and oxygen. To support this massive claim of energy and oxygen the

cerebral blood vessel network is enormous. The blood flow is rapidly increased at sites

of activity in the brain to accommodate the high energy demand, this is known as

neurovascular coupling (1). Brain endothelial cells make up the micro-vessels of the

brain and have features that differentiate them from endothelial cells in other organs.

Brain endothelial cells have longer continuous stretches of TJs, higher number of

mitochondria, no fenestrae (small pores) and low pinocytic activity (12-14). All of

these features contribute to the brain endothelial cell capacity to restrict permeability

and act as a selective barrier. TJs are important structures in brain endothelial cells that

separate the blood face from the brain face of the cells. TJ structure and function are

further discussed in section 2.1. The different faces of the endothelial cells have

distinct properties, making endothelial cells polarized. TJ restriction of water-soluble

molecules in the paracellular space cause high trans-endothelial electrical resistance

(TEER), a hallmark of brain endothelial cells. Physiological brain TEER is estimated

to be above 1000Ohm x cm2 compared with 2-20Ohm x cm2 in the majority of the

body (15). TJ proteins, such as claudin-5, occludin and specific transporters, such as

P-glycoprotein (P-gp) and Glut-1 are often used as markers of brain endothelial cells.

The study of brain endothelial cells has been hampered by the difficulty to obtain

human primary brain endothelial cells from healthy individuals and the fact that human

primary brain endothelial cells and immortalized bran endothelial cell lines do not

maintain barrier restriction capacity in vitro (2). Primary endothelial cells isolated from

animals such as pigs and rats retain fairly tight barriers in vitro and can be useful tools

to study paracellular permeability (2, 16). However, the restrictive capacity in vivo and

the expression of specific transporters are different between species (17, 18). Hence,

to be able to predict and study the human BBB a human model is highly preferable.


5

Astrocytes

Astrocytes or astroglia are at least as abundant as neurons in the adult human brain

(19), and the number of astrocytes has even been speculated to be one of the features

explaining human cognitive abilities (4). Astrocytes are a very diverse cell type, both

morphologically, molecularly and functionally (20). During development of the human

brain, astrocytes in the cerebral cortex are derived from four distinct progenitor

populations: radial glia, subventricular zone progenitors, locally proliferating glia and

neuron glia antigen 2 (NG2) glia. To what extent each of these four progenitor

populations contribute to the production of glia remains elusive. Although, it is clear

that each of these subpopulations of progenitors contributes to the production of

astrocytes at distinct stages of development and at different locations (19). The

specifics of the astrocytic developmental process are not yet fully understood. One of

the underlying reasons for this lack of understanding is the absence of well-

characterized markers to study astrocytes and their progenitors during development.

Specific functional and molecular profiles of astrocytes depend on signalling from

surrounding cells, astrocytic diversity and heterogeneity are defined by both regional

identity and functional input from surrounding neurons (21). Microarray analysis has

revealed a small number of genes that are expressed by most astrocytes, while

expression of other genes is specific to astrocytes in certain brain regions (22).

The understanding of astrocyte functions has evolved over time from being considered

as a passive helper cell to the insight that astrocytes play a crucial role in development,

maintenance and aging of the CNS. Astrocytes are key players in the CNS and one

astrocyte domain can contain many million synapses (20). Astrocytes regulate the

formation of synapses, control synaptic signalling (23), and support rapid and accurate

synaptic signalling by regulating availability of nutrients and neurotransmitters (24,

25). Glutamate is the most abundant excitatory neurotransmitter. Glutamate uptake by

astrocytes serve as a protective mechanism for excitotoxicity in adjacent neurons

(25). By removing excess glutamate from the synaptic cleft, the astrocytes maintain

homeostasis and signalling fidelity in the synapse. Glutamate is taken up, mainly,

via the EAAT1 and EAAT2 transporters and is then converted to glutamine by

glutamine synthetase (GLUL) (Figure 2). Glutamine can subsequently be exported

from the astrocyte via the glutamine exporters SNAT3, SNAT5, ASCT2 and be

reused by the neurons. Glutamate uptake does not only serve as a protective

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mechanism for neurons but also activates glycolysis and glucose uptake in

astrocytes.

Figure 2. Glutamate metabolism. Excess glutamate (GLU) in the synaptic cleft is

taken up into the astrocyte by EAAT1 and EAAT2. In the astrocyte, glutamate is

converted to glutamine (GLN) by glutamine synthetase (GLUL). Glutamine can then

be transported out of the astrocyte by glutamine exporters SNAT3, SNAT5 and

ASCT2 and be reused in the neuron.

Through their end-feet, astrocytes are in close proximity to brain endothelial cells and

affect the development and maintenance of the BBB through physical interaction and

secretion of signalling molecules (26, 27). Astrocytes produce a range of molecules

that are associated with the BBB phenotype and proper differentiation of brain

endothelial cells, including components of the BM, glia-derived neurotrophic factor

(GDNF), VEGF, apolipoprotein E (apoE), transforming growth factor beta (TGF-β),

inter leukin 6 (IL6) and angiopoietin 1 (Ang-1) (1, 27). Furthermore, SHH produced

by astrocytes cause upregulation of TJ components and reduced permeability in brain

endothelial cells (10). Astrocytes also help regulate the blood flow within the brain

through calcium signalling in their end-feet (28). Many pathological changes in the

CNS are accompanied by astrocyte activation, commonly identified by increased cell


7

body volume and upregulation of intermediate filament proteins, such as glial fibrillary

acid protein (GFAP) and vimentin (29). Activation of astrocytes and subsequent

production of cytokines and pro-inflammatory substances in the brain can be

protective, however, there is evidence that prolonged activation has detrimental effects

in many CNS trauma and neurodegenerative conditions (29). The production of

cytokines and inflammatory molecules from activated astrocytes are likely to influence

the closely connected brain vasculature and thus the BBB. Reactive astrocytes have

been suggested to increase secretion of both BBB promoting and disrupting factors

(10, 30). However, the understanding of how reactive astrocytes influence the BBB in

vivo is still poor.

Pericytes

There is general consensus that pericytes are a highly diverse cell type with different

subtypes having different functions and characteristics. Interestingly, while most

pericytes are believed to be of mesodermal origin, studies in birds have suggested that

CNS pericytes derive from the neural crest (31). Due to the ambiguity of universal

pericyte properties, finding a gold standard protein marker to identify these cells has

been challenging. As such, the identification of pericytes relies on a combination of

morphological criteria and assessment of marker expression. Expression of proteins,

such as platelet derived growth factor receptor beta (PDGFRβ), NG2, caldesmon,

CD13 and CD146, is commonly used to identify pericytes. Characterization of

different subtypes of pericytes are underway. Vitronectin and fork head transcription

factors FOXF2 and FOXC1, have been shown to be expressed specifically in brain

pericytes (32-34). In addition to their brain pericyte specific expression, FOXF1 and

FOXC1 have been shown to influence BBB development.

At the BBB the pericytes ensheath the endothelial cells. Pericytes are believed to play

a specific role in the neurovasculature, as neural tissue has higher pericyte coverage of

the vasculature compared with other organs. Neural tissue has up to one pericyte per

one endothelial cell (35). Additionally, pericyte coverage correlate positively with

endothelial barrier properties of different tissues. Pericytes have a diverse set of

functions, they aid in angiogenesis and microvasculature stabilization, regulate

capillary diameter and phagocyte toxic compounds (35). Pericytes produce many of

the BM components and in that way contribute further to the structure of the BBB (36).

Large parts of the current knowledge around pericyte function come from studies in

pericyte-deficient rodents. These rodents show a number of BBB abnormalities, such

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as increased permeability, increased transcytosis and irregular TJs (11). Studies show

that developing endothelial cells attract pericytes by PDGFβ-signalling, leading to

pericyte proliferation and co-migration with endothelial cells (11). Pericytes then

contribute to brain endothelial cell specific maturation such as formation of TJs,

reduced vesicle trafficking and reduced permeability (11). Human in vivo studies have

shown increased cerebrospinal fluid (CSF) markers of pericyte damage and BBB

breakdown in cognitive impairment patients compared to healthy individuals (37). In

summary, pericytes play an essential role in both BBB formation and maintenance, but

many of the mechanisms behind pericyte influence on the BBB are still unknown.

Neurons and microglia

Both neurons and microglia affect the BBB, however their contributions are less

studied than those of pericytes and astrocytes. Microglia are the innate immune cells

of the brain. Despite their name and stellate morphology, they do not share the common

neural stem cells origin with neurons, astrocytes and oligodendrocytes. Mouse studies

suggest that microglia progenitors originate from the yolk sack and migrate into the

CNS during early embryogenesis, later, these cells proliferate to populate the CNS

with microglia (38). Microglia invasion of the CNS precedes vascular sprouting into

the tissue, but when vessels appear, endothelial cells and microglia are in close

proximity to each other. Microglia have been suggested to play a role in both

endothelial cell stability and angiogenesis during the development of the brain

vasculature (39). Microglia become activated in response to injury and immunological

stimulation. Active microglia can affect BBB stability and increase the permeability

across the BBB. Studies of rodent in vitro models have suggested that these effects

depend on reactive oxygen species (40) and tumour necrosis factor alpha (TNFα) (41)

released by activated microglia. Microglia are important players in BBB formation and

maintenance in vivo, however, as immunological challenges are kept to a minimum in

in vitro BBB models, microglial contributions were not investigated in this thesis.

Neurons make up the main signalling networks of the brain. Neuronal signalling

requires large amounts of energy and signalling from the neurons affects the blood

flow through the brain vasculature to ensure enough energy supply. TJ stabilization

was observed in cocultures of brain endothelial cells and neurons. Brain endothelial

cells in the coculture were also induced to synthesize and sort occludin to the surface

(42), indicating that the stabilization of TJs in coculture with neurons may be an effect

of increased occludin production. Neurons and astrocytes are tightly coupled,


9

astrocytes support correct signalling environment for neurons and neuronal presence

aids in astrocyte maturation (21, 23). Hence, the effects that neurons have on the BBB

are likely to be both direct signalling from the neurons to the endothelium and

secondary effects that arise via changes that neurons promote in the astrocytes. In this

thesis, neuron cocultures were performed in the in vitro BBB models, however their

specific contributions were not examined in detail.

The basement membrane

The BM is the non-cellular component of the BBB, it is a specific extracellular matrix

that surrounds the endothelial cells. The BM contains highly conserved proteins, and

consists mainly of laminin, collagen IV, perlecan, and nidogen (43, 44). The BM

contributes to structural support and signalling by binding growth factors and

neurotrophic factors such as fibroblast growth factor (FGF), VEGF and GDNF (27,

45, 46). Endothelial cells, pericytes and astrocytes secrete the proteins, which together,

make up the BM (36, 44, 47, 48). Laminins are the most abundant component of the

BM. In addition to their structural functions, laminins play an essential role in the

organization of the BM and the regulation of cell behaviour (47-49), hence the BM

modulation in this work has focused on laminins. Laminins are multidomain,

heterotrimeric glycoproteins, composed of three different subunits; an α-chain, β-chain

and γ-chain, combined and expressed in at least 16 different isoforms in the human

body (50). The physical, topological, and biochemical expression of the different

laminin isoforms in the BM is heterogeneous and laminin expression changes during

development. Without the right combination of laminin isoforms, cells and tissues

become dysfunctional. Brain endothelial cells generate laminins 411 and 511 (47)

whereas astrocytes produce laminins 111, 211 (48). Furthermore, mouse studies

suggest that laminin alpha 5 is more highly expression in brain than in the periphery

and that it is important in protecting the brain vasculature from mononuclear

infiltration (47). Laminin 521 has been shown to be specifically important for astrocyte

migration and vascularization in the retina (51). All the above laminin isoforms are

also expressed by the primary brain capillary pericytes (47, 49, 52). Effects of culture

on de-cellularized ECM from pericytes, astrocytes and brain capillary endothelial cells

on brain endothelial cell differentiation from iPSC was recently investigated (53). It

was concluded that the de-cellularized ECM from astrocytes had the most beneficial

effects on brain endothelial cell differentiation, however, this was not significantly

different from using fibronectin. iPSC-derived brain endothelial cells did not adhere to

Delsing, Louise

10

de-cellularized pericyte ECM suggesting that ECM produced by pericytes alone is not

sufficient to support brain endothelial cell cultures. However, the de-cellularized ECM

used in these experiments were derived from animal sources and it cannot be excluded

that ECM from human sources would give a different result. The BM is a complex

mixture of ECM from several cell sources and synergistic effects may be at play in the

BM that are not accurately modelled using ECM from individual cell types.

Consequently, the BM is highly important for normal BBB formation and function,

but it remains unclear exactly how individual components contribute.

The blood-brain barrier and disease

This thesis focuses on modelling of the healthy BBB. However, to appreciate possible

future applications for iPSC-derived BBB models it is helpful to understand how the

BBB is linked to disease. The BBB is of major clinical relevance as dysfunction of the

BBB is observed in many neurological diseases, and the efficacy of drugs designed to

treat neurological disorders is often limited by their inability to cross the BBB. BBB

disruption is observed in many pathological conditions such as, stroke, multiple

sclerosis (MS), human immunodeficiency virus (HIV) encephalitis, and Alzheimer’s

disease (AD) (12), both as a cause of disease and as a symptom. BBB disruption refers

to a decrease in the tightness of the barrier, resulting in less controlled transport across

the barrier and higher permeability from the blood into the CNS. The connection

between BBB permeability and AD is of increasing interest as the BBB is suggested

to play a role in the accumulation of the AD hallmark amyloid β (Aβ) peptides. It has

been hypothesized that a deficiency in the efflux of Aβ from the CNS contributes to

accumulation and toxicity (54). This has also been shown in mouse models with

mutations known to cause age-dependent Aβ accumulation and cognitive impairment

(55). Efflux of Aβ is suggested to be mediated by the low-density lipoprotein receptor-

related protein 1 (LRP-1). In a mouse model, where LRP-1 was knocked down by

antisense, brain Aβ levels increased by 40% and cognitive function declined. In

addition, efflux of Aβ1-42 was more significantly lost than efflux of Aβ1-40, favouring

retention of the more toxic form with aging (55). There is an increased uptake of Aβ

into pericytes in AD and Aβ overload could explain the loss of pericytes seen in AD.

Mouse models suggest that pericyte loss influences disease progression in AD by

diminishing clearance of Aβ (56).

Another example of a neurological disease with prominent BBB contributions is MS

(57). MS is initiated by activation of myelin autoreactive T-cells that drive


11

inflammatory response against myelin antigens in the CNS. In response to the

inflammation, several pathways leading to loss of BBB tightness are activated, for

example metalloproteases degrading the ECM and basement membrane. As BBB

integrity is compromised, T-cells infiltrate the CNS and there is activation of

macrophages and microglia. This collectively leads to demyelination, plaque

formation, and ultimately neurodegeneration. Clearly, the BBB plays a major role in

several diseases and consequently therapeutic targeting of the BBB is likely to

increase.

The blood-brain barrier in drug discovery

Diseases of the CNS are affecting an increasing number of individuals worldwide as

the populations of most countries are aging. Furthermore, for many common diseases,

such as neurodegenerative disorders, autism spectrum disorders and schizophrenia,

there are no treatments affecting the disease and only few and inadequate options for

treating the symptoms (58). The unmet medical need in neurological disorders is

significant. A contributing factor is the many challenges in drug development of CNS

active drugs. It has been estimated that almost all large molecule therapeutics and the

majority of all small molecule drugs fail to penetrate the BBB (59). At the same time,

major pharmaceutical companies are decreasing their investment in neuroscience

research compared to other therapeutic areas (58). One plausible reason for the

decrease in investment is the disproportionate failure rate in later stage clinical trials

for CNS-targeting drugs compared to non-CNS-targeting drugs (60). Major challenges

in CNS drug discovery include the multifactorial nature of many CNS diseases,

difficulties in modelling the complexity of the CNS in vitro, and predicting BBB

permeability. Consequently, there is a need for reliable models of the human BBB with

high precision predictions of BBB permeability of novel drug molecules.

Delsing, Louise

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13

2 PERMEABILITY OF THE BLOOD-BRAIN BARRIER

Controlled movement across the BBB involves restriction and facilitated transport of

essential substances to supply nutrients. Transport into the CNS occurs through

paracellular transport, transcellular diffusion, carrier-mediated-transport (CMT),

receptor-mediated-transcytosis (RMT) and transcytosis (Figure 3). In addition, ATP-

dependent efflux transporters and ion pumps are active at the BBB. Small hydrophilic

molecules may pass through the paracellular route; however, due to the high density

of TJs in brain endothelial cells, this transport route is very restricted. Oxygen and

carbon dioxide freely diffuse across the endothelial cell membrane in transcellular

transport. Similarly, small lipophilic molecules, such as ethanol, can diffuse across.

Figure 3. Transport across the blood-brain barrier. Small molecules such as oxygen

and carbon dioxide can diffuse across the endothelial cell membrane in transcellular

diffusion. Selective mediated transport occurs via receptor-mediated-transcytosis

(RMT) or carrier-mediated-transport (CMT). In RMT the transported substance

binds to a receptor which is subsequently internalized in a vesicle, transported

across the cytosol and released on the other side of the cell by fusion of the vesicle

with the membrane. CMT-specific transporters allow substances to pass through the

cell down their concentration gradient. Diffusion in the paracellular space is very

restricted at the BBB due to the high density of tight junctions, however, some

molecules can still pass the barrier via paracellular diffusion. Transcytosis occurs

via endocytosis, transport across the cell and exocytosis similar to RMT but without

depending on receptors to bind the transported molecules. Efflux transport is

polarized and occurs through pumping of substances from the cytosol back into the

blood.

Delsing, Louise

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Larger molecules and nutrients such as glucose and amino acids rely on CMT or RMT.

Moreover, the BBB is polarized, which means that the blood-facing side and the brain-

facing side of the endothelial cells have different compositions of transporters. The TJs

between the endothelial cells function as boundaries restricting diffusion of

transporters between the blood and the brain sides of the endothelial cells, maintaining

the polarization of transporters.

Inter-cellular junctions

Inter-cellular junctions between the endothelial cells at the BBB are made up of TJs

and adherens junctions (AJs), in addition cluster of differentiation 31 (CD31) protein

is highly expressed and its connections contribute to cell-cell adhesion (Figure 4) (61,

62). Very constricted TJs are a hallmark of the BBB and limit the permeability of polar

solutes in the paracellular space. AJs connect the cells through transmembrane

cadherins, which reach from the cytoplasm to the extra-cellular space between the

cells. Cadherins are linked to the cytoskeleton by the scaffolding proteins alpha-, beta-

and gamma catenin. In brain endothelial cells, VE-cadherin is the most prevalent

cadherin with only low levels of E and N-cadherin (62). The composition of TJs is

more complex; transmembrane proteins; occludins, claudins and junctional adhesion

molecules (JAMs) span the junctions between the cells. Occludins and claudins are

linked to the cytoplasmic scaffolding proteins zonula occludens-1, 2 and 3 (ZO-1, ZO-

2, ZO-3). The claudin family is large and diverse, with more than 20 known subtypes

(63). Claudin-5 is commonly identified as the most abundant claudin in brain

endothelial cells (62), and both claudin-5 and claudin-3 have been shown to localize

at TJs in the brain endothelium (64). WNT-signalling has been suggested to play an

important role in stabilizing TJs and enhancing barrier properties, at least partly,

through the regulation of claudin-3 (65, 66). The TJs are responsible for restricting the

permeability of soluble molecules and ions, which gives rise to the high electrical

resistance often used to characterize highly impermeable cell layers. In addition to

anchoring the claudins and occludins to the cytoskeleton, ZO-1, ZO-2 and ZO-3

function as regulatory elements by interacting with cytoplasmic proteins, signalling

molecules and transcriptional regulators (67). Occludin expression is higher in brain

endothelial cells than in peripheral endothelial cells and occludin expression levels

have been shown to correlate with barrier tightness (2, 68, 69). The importance of

occludin in the brain vasculature is further reinforced by reports of rare mutations in

the occludin gene causing a disorder with severe malformations in cortical


15

development (70). TJ-associated proteins play an important part in creating the unique

phenotype of brain endothelial cells and are frequently used to identify and visualize

brain endothelial cells.

Figure 4. Inter cellular junctions. Tight junctions contain occludins, claudins and

JAMs which span the paracellular space. These are linked to the cytoskeleton via

zonula occludes. Adherens junctions contain cadherins which span the paracellular

space and are linked in the cytoskeleton via catenins. In addition, CD31 contributes

to intercellular connections.

Transport proteins

Brain endothelial cells control the ability of molecules and ions to diffuse from the

blood into the brain. To supply the brain with required energy and nutrients, specific

proteins at the BBB facilitate the transport (Figure 3). Selective mediated transport

occurs through either CMT or RMT. CMT enables molecules such as carbohydrates,

amino acids and vitamins to be transported down their concentration gradient through

membrane carrier proteins. Examples of CMT include the solute carrier (SLC)

transporters and the amino acid transporters (LATs). The energy supply to the brain is

facilitated in this manner; the SLC transporter Glut-1 transports glucose down its

concentration gradient from the blood into the CNS (71). In addition to Glut-1, the

SLC transporter family contains numerous other transporters essential to the BBB,

such as nucleoside and peptide transporters. RMT mediates transport of proteins and

peptides through the binding of these to specific receptors. The receptors are

subsequently internalized with the protein or peptide attached, shuttled across the

Delsing, Louise

16

cytoplasm and released on the other side. RMT is responsible for the transport of

nutrients and hormones such as iron, leptin and insulin across the BBB. Clathrin and

caveolin mediate the formation of vesicles for RMT and non-receptor-mediated

vesicular transport (72). Reduced caveolin-mediated transport has been identified as a

differential factor between brain endothelial cells and peripheral endothelial cells.

Furthermore, increased caveolin vesicle transport has been implicated as a contributing

factor to barrier leakage (32, 73). As caveolin, but not clathrin, has been identified as

a differential factor in BBB permeability, caveolin expression level is commonly

investigated as an indicator of vesicular trafficking.

Efflux transporters

The efflux transporter system works as a second security mechanism in the control of

BBB permeability. Some substances may be able to diffuse across the cell membrane

or are able to pass into the cell through CMT. However, they will have substantially

reduced permeability into the CNS if they are recognized by the efflux transporters.

Substrates of efflux transporters are efficiently shuttled back into the blood. Efflux

transporters are ATP-binding cassette (ABC) transporters, they hydrolyse ATP to

provide energy for transport of substances across the blood-side endothelial

membrane. The three main efflux transporters are P-glycoprotein (P-gp), breast cancer

resistance protein (BCRP) and multidrug resistance proteins (MRPs) (18). Efflux

transporters have a broad substrate range, particularly P-gp, and are responsible for the

low permeability into the CNS of many endogenous and exogenous molecules

circulating in the blood. This protects the CNS from substances such as xenobiotics,

pesticides and drugs, that could be harmful to the brain. Chemical properties of many

drug molecules allow them to diffuse across cell membranes, however, they are also

common substrates for efflux transporters, reducing their transport across the BBB

(74).

Assessing BBB permeability of novel drug candidates is of high importance, both for

drugs targeting the CNS and other organs. For drugs targeting the CNS, there is a need

to assess if penetration of the BBB is sufficient for the drug to be active and reach its

target in the CNS. For example, many anti-cancer agents have very limited effects on

cancers of the CNS due to low penetration of the BBB (75). On the contrary, a drug

with a target outside of the CNS should preferably not enter the CNS as that may cause

additional side effects. For example, the beta-blocker propranolol has been shown to

readily cross the BBB and generate side effects, such as hallucinations, nightmares and


17

sleep disturbance, at a higher incidence than other beta-blockers with lower BBB

permeability (76). Furthermore, it is important to evaluate drug-drug interactions,

which can cause one drug to affect the clearance of another drug. One common way

through which this occurs is when a drug affects efflux transporter activity.

Interactions between drugs need to be carefully evaluated in drug discovery, and

guidelines for how to perform these evaluations have been suggested by the US Food

and Drug Administration (FDA). One of the key recommendations for evaluating drug-

drug interaction is to investigate if the drug has interactions with efflux transporter

proteins P-gp and BCRP. The expression levels of BCRP and P-gp are substantially

higher than expression of MRPs in brain endothelial cells (62). Hence, evaluation of

efflux activity in the models developed in this thesis has focused on P-gp and BCRP.

Clinically significant interactions with P-gp and BCRP have been reported for several

drugs. For example, digoxin, which is used to treat various heart conditions, has been

shown to interact with P-gp and is often co-administered with other P-gp interacting

substances. Upon co-administration the availability of digoxin changes to correspond

to a higher intake, which results in increased risk of over-dosing and generating side

effects (77). Furthermore, at the BBB, common antidepressants such as selective

serotonin re-uptake inhibitors, have been suggested to reduce the activity of P-gp,

hence reducing its capacity to act as a protector of the CNS. Consequently, efflux

transporter interaction studies are highly important in predicting BBB permeability and

side effects of novel drug candidates.

Modulating blood-brain barrier transport for therapeutic purposes

Entry into the CNS is a major challenge for many novel drug substances and one of

the major hurdles in drug development for neurological disorders. Studies of how

pathogens enter the CNS have revealed that interaction with surface proteins on the

brain endothelial cells facilitate the process. For example, E. coli interacts with a

glycoprotein on the brain endothelial cell surface, which facilitates its penetration of

the BBB (78). This observation provoked ideas of adopting similar strategies for drug

delivery. Several approaches to increase the permeability of drugs to the CNS are under

investigation. Current strategies include transient opening of the BBB and using drug

carriers or ligands that facilitate penetration (79, 80). For example, cancer drugs have

been linked to amino acid sequences, recognized by the RMT system, to increase their

BBB permeability (79). Furthermore, exploiting the RMT system can be used to target

Delsing, Louise

18

specific regions of the brain with high expression of certain receptors. Other strategies

have focused on reducing the activity of efflux transporters, specific inhibitors, such

as elacridar and tariquidar, have been developed. Clinical trials to increase CNS

availability of efflux transporter substrates using these inhibitors are ongoing (81).

Modulating BBB permeability may be beneficial in increasing permeability of some

drugs to the CNS, however, changing the general permeability of the BBB may have

very severe side effects. Selective modulation of BBB permeability is preferable but

clearly more difficult to accomplish.


19

3 iPSC-DERIVED CELLS FOR BLOOD-BRAIN BARRIER MODELING

Induced pluripotent stem cells (iPSC) are somatic cells reprogrammed to a pluripotent

state using overexpression of defined transcription factors (82) (Figure 5). iPSCs are

similar to embryonic stem cells (ESC) and can be differentiated to most cell types of

the human body. iPSCs do not suffer from the same ethical obstacles as ESCs because

they can be generated from cells obtained from an adult individual. The generation of

iPSCs from adult cells allows for a number of new applications. Some cell types, such

as neural cells and brain endothelial cells, are difficult to study in vitro due to the

challenges in obtaining these cells from healthy individuals.

Figure 5. Induced pluripotent stem cells are reprogrammed adult human cells from

patients or healthy individuals. Once reprogrammed to an early development stage

induced pluripotent stem cells can self-renew and be differentiated into most cell

types of the human body.

The iPSC technology provides great possibilities to generate large amounts of these

cell types for in vitro studies without invasive sampling of healthy humans or use of

animals for research purposes. Furthermore, iPSCs can be generated from patients to

provide patient-specific cell lines, which can be used to produce and regenerate

damaged tissues. iPSCs have a high proliferation capacity making them suitable for

Delsing, Louise

20

large scale production of cells as well as genetic manipulation. To unleash the potential

of iPSCs, robust and reliable protocols for differentiation are required. The

development of differentiation protocols for directing iPSCs to a specific cell type

generally relies on recreating the signalling processes that govern the development of

the desired cell type during embryogenesis.

iPSC-derived endothelial cells

Among well-defined signalling pathways, bone morphogenetic protein (BMP), FGF,

and VEGF-signalling are most commonly modified for endothelial differentiation

(83). The BMP family modulates early vascular development via the downstream

SMAD family proteins, as demonstrated by studies in human embryonic stem cells

(84). Treating stem cells with BMP early in the differentiation process has been shown

to significantly induced endothelial differentiation (85). Notably, the VEGF family

members were among the first secreted molecules observed to be specific to

endothelial differentiation. VEGF receptors that are specific to the endothelial lineage,

contribute to endothelial differentiation (86). This suggests that VEGF is not an early

endothelial signalling cue but rather a later specification factor.

The brain endothelial cells have different properties than the peripheral endothelial

cells. Hence, the differentiation of brain endothelial cells from iPSCs may require

specialized protocols different from those used to derive peripheral endothelial cells.

In 2012, Lippman et al. published a protocol for differentiation of iPSCs to brain

endothelial cells (87). During the years after 2012 several improvements of the

protocol have been proposed, including the addition of retinoic acid (RA) (88),

optimizing of seeding density (89) and use of more defined medium components (90-

92). The protocol relies on spontaneous co-differentiation of endothelial cells with

neural cells and subsequent purification of the endothelial cells by passage on-to

collagen/fibronectin in an endothelial cell medium containing FGF and RA.

Particularly the RA treatment at the end of the differentiation has proven important for

the cells to develop a mature BBB phenotype, with high tightness and increased

expression of several TJ proteins and transporters (88, 91). Endothelial cells generated

with this protocol display high TEER 500-4000Ohm x cm2, low permeability and

expression of claudin-5, occludin, ZO-1, CD31, VE-cadherin and Glut-1 (88, 90, 93).

Most recently, fully defined versions of this protocol that eliminate the use of serum

have been proposed. These protocols give similar results as the original versions and

rely on sequentially activating WNT- and RA-signalling, or spontaneous


21

differentiation followed by RA-signalling (91, 92). Other protocols for derivation of

brain endothelial cells have been proposed, but without successful adoption in the iPSC

BBB community. A proprietary method for deriving brain endothelial cells has been

used in an investigation of apoE4 mediated endothelial cell toxicity, and more recently

in a self-organizing microphysiological model (94, 95). Other protocols have used

directed differentiation by sequential BMP and VEGF treatment to initiate

differentiation to endothelial cells. Although these cells displayed upregulation of

brain endothelial cell markers, they did not form tight monolayers and showed TEER

values of approximately 50Ohm x cm2 (53, 96). The protocol developed by Lippmann

et al., and subsequent optimizations of it, remains the most widely used methods for

derivation of brain endothelial cells from iPSCs for in vitro BBB models.

iPSC-derived pericytes

The development of differentiation protocols for pericytes has been hampered by the

lack of detailed knowledge of the pericyte characteristics. Pericyte marker proteins,

functional characteristics and even their origin have been debated. Before brain

pericyte-specific protocols were developed, pericytes were mainly differentiated

through mesodermal intermediates. Differentiation of the pericytes used in this thesis

was performed before brain-specific pericyte differentiation protocols were developed.

As such, the pericyte differentiation approach used in this thesis depends on

mesodermal induction and further differentiation of sorted cells that lack CD31

expression (97). Mesodermal differentiation was induced through activating TGF-β

signalling by BMP4 and Activin, activation of WNT signalling and VEGF treatment,

subsequent vascular specification through inhibition of TGF-β-signalling and

continued VEGF treatment. After sorting of CD31-positive cells, the CD31-negative

cells were further differentiated to pericytes by treatment with FBS, PDGFβ and TGF-

β. These pericytes were characterized through expression of caldesmon, SM22 and

smooth muscle actin. Recently, an in-depth analysis of the cell types in the brain

vasculature has provided new insight into the brain pericyte phenotype, and new

markers that differentiate brain pericytes from peripheral pericytes were proposed,

such as a higher abundance of SLC, ABC and ATP transporters (62). Brain pericytes

have been shown to develop from neural crest stem cells (31), and recently a protocol

for derivation of brain pericytes from iPSCs via neural crest stem cells was published

(98). Initiation of neural crest differentiation was performed by WNT activation and

TGF-β inhibition, neural crest stem cells were enriched through sorting, and pericytes

Delsing, Louise

22

were generated through serum treatment. However, pericyte differentiation through

both neural crest stem cells and mesodermal intermediates has given similar results

(99). Mesodermal pericytes were derived via mesodermal induction and subsequent

pericyte differentiation through culture in a proprietary medium promoting pericyte

growth. Neural crest pericytes were derived through activation of WNT to derive

neural crest cells and subsequent culture in the same pericyte growth medium.

Development of well-defined differentiation protocols to derive brain pericytes from

iPSCs is still in its initial stages. However, recent developments in generating brain-

specific pericytes hold great promise for their use in iPSC-derived BBB models. iPSC-

derived brain pericytes have been shown to reduce permeability of iPSC-derived brain

endothelial cells at similar levels as induction by astrocytes and neurons (98). Since

the role of pericytes may be primarily structural it is possible that Transwell BBB

models benefit less from pericyte cocultures than microfluidic models where

endothelial cells form vessel-like structures.

iPSC-derived astrocytes

There are several published protocols for astrocyte differentiation from iPSCs, and

iPSC-derived astrocytes have been used to study many different aspects of astrocyte

biology including inflammatory response (100-102), glutamate uptake (101, 103),

apoE biology (104) and genome wide expression studies (100, 101). Indeed, iPSC-

derived astrocytes have proven to be a powerful tool for understanding human

astrocyte biology in health and disease. Astrocyte development and maturation occurs

late in the embryonic development and continues after birth (4). As such, mimicking

the in vivo astrocyte development is a very lengthy process, often spanning several

months. Many protocols for astrocyte differentiation rely on long-term culture of

neural stem cells in FGF and epidermal growth factor (EGF) and/or serum (105, 106).

iPSC-derived astrocytes are commonly characterized by their expression of GFAP,

CD44, EAAT1/2, S100B, and vimentin, and their ability to perform astrocyte specific

tasks, such as glutamate uptake and inflammatory response to treatment with

inflammation regulators (103, 106). Long-term differentiation protocols often require

repeated passaging, selecting the proliferating population, which is likely to contribute

to the long differentiation time as maturation of astrocytes generally reduces

proliferation. There have been numerous efforts to shorten the differentiation time

required for astrocyte development, for example, through remodelling of the chromatin

structure (107) and using genetic techniques to overexpress transcription factors SOX9


23

and NF1A that govern the gliogenic switch in which neural stem cells switch from

neurogenesis to gliogenesis (108).

In this thesis, neural stem cells were derived using spontaneous differentiation and

manual isolation of neural rosette forming cells (109). These were subsequently

cultured in FGF and EGF and maintained as long-term neuroepithelial stem cells (lt-

NES) expressing the neural stem cell markers SOX1, SOX2 and Nestin. The protocol

used to derive astrocytes from lt-NES is a slightly modified version of the protocol

first published by Shaltouki et al. (103). It relies on 4 weeks culture of neural stem

cells in FGF, heregulin, insulin-like growth factor 1 (IGF1) and activin A to drive

astrocyte differentiation. It is still unclear exactly how these factors drive astrocyte

differentiation, however, heregulin has been shown to play a particularly important

role. Heregulin is a splice variant of neuregulin. It interacts with the EGF family of

receptors and is able to induce astrocyte differentiation (103). Even with recent efforts

to shorten protocols for astrocyte differentiation the process is still labour-intensive

and long. Furthermore, the understanding of heterogeneity in human astrocytes is

increasing and there is a growing interest in generating subtype-specific astrocytes

from iPSCs. Both major astrocytic subtypes, protoplasmic and fibrous astrocytes, are

in contact with the blood vessels in vivo (20). However, it remains unknown if

astrocyte subtype influences the effects that astrocytes have on the brain vasculature

and the BBB.

Delsing, Louise

24


25

4 BLOOD-BRAIN BARRIER MODELS

Models of the BBB serve as important tools in drug development and support

evaluation of chemical properties and brain penetrating capacities of novel drug

molecules. Regardless if the brain is the intended target or not, it is central to

understand the permeability of a drug candidate into the CNS. Although many drug

candidates appear promising in animal models, as many as 80% of them later fail in

clinical trials (110). This clearly demonstrates the need for better pre-clinical models

with higher translatability to the human in vivo situation. At the same time, large

efforts are being made to reduce the use of animal testing in research. The three Rs

ethical principle to reduce, replace and refine animal-based science is widely accepted

and implemented throughout the research community. In many countries, the three Rs

principle is explicit legislation. Human cell-based models are important alternatives to

in vivo animal models and models using animal cells.

Current models of the BBB span from in vivo animal models to more complex

cocultures of several primary cell types and in silico modelling (111, 112). In vivo

animal models, using techniques such as brain perfusion, are considered some of the

most accurate ways of determining BBB penetration. However, these techniques

require animals to be subject to research, are time consuming, expensive and have low

throughput, compared to cellular models (111). A wide range of cellular models of the

BBB have been described, including primary cells and cell lines from both human and

animal origin. Primary cells from animals have proven to have suitable barrier integrity

and relatively low permeability (112, 113), but disadvantages linked to the use of

animal cells include resource demanding isolation procedures, batch-to-batch

variability and incompatibility with reducing the use of animals for research. An

important aspect of BBB modelling using animal cells is the differences between the

human BBB and the BBB in other species. For example, there is evidence of species

differences in the expression of BBB transporters, including the important efflux

transporter P-gp, and in permeability of P-gp substrates (17, 18). By using

immortalized cell lines from both human and animal origin, issues with reproducibility

and batch variability can be circumvented. However, many of the immortalized brain

endothelial cell lines available fail to form tight barriers with low permeability, which

questions their usability.

Availability of primary human brain cells is very limited, and samples are typically

residual tissue from patient biopsies or post mortem brains. In addition, isolated

Delsing, Louise

26

primary brain endothelial cells rapidly lose their BBB properties when cultured in vitro

(2). Considering this loss of functionality in vitro, it is plausible that the BBB

properties are not intrinsic to the brain endothelial cells but rather depend on the

specific microenvironment that all components of the NVU create together. This

suggests that a more complex model with coculture of multiple cell types is needed to

recapitulate important functions of the BBB. Several coculture models have been

described that demonstrate improved barrier properties compared to endothelial cells

alone. There is a need for models that recapitulate the combined barrier functions of

the BBB. A reproducible model from a continuous human cell source would increase

the possibility of the model to be used in high-throughput screening experiments.

A recent review identified five groups of criteria for benchmarking in vitro BBB

models; structure (ultrastructure, wall shear stress, geometry), microenvironment

(basement membrane and extracellular matrix), barrier function (TEER, permeability,

efflux transport), cell function (expression of BBB markers, turnover), and coculture

with other cell types (astrocytes and pericytes) (114). Historically, in vitro BBB models

have used static two-dimensional (2D) cultures in Transwell plates, but recently

perfused three-dimensional (3D) models have gained increasing interest (Figure 6). 2D

static models have been very useful in providing new understanding of mechanisms of

transport across the BBB and are still widely used to assess CNS transport in drug

discovery processes. In 3D models the structure of the brain vasculature can be more

accurately modelled and flow can be added to introduce shear stress, which more

closely recapitulates the in vivo conditions. Even though most of our knowledge about

BBB in vitro models come from static systems, shear stress arising from flow is

thought to regulate several BBB specific properties, for example permeability,

metabolism and expression of transporters (115). In an in vitro vessel with cylindrical

geometry, there are relatively few cells that form tight interactions with each other

around the vessel. This limits the motility of the cells more than in the 2D sheet of cells

present in the Transwell models, and more accurately reflects the in vivo conditions.

The basement membrane generates a specific microenvironment for the brain

endothelial cells and its composition has been shown to effect BBB specificity of

endothelial cells (36, 43, 44, 53). ECM proteins, such as collagen 1, are widely used

in in vitro models but are not present at the healthy human BBB. Consequently, in vitro

models need to provide a mix of ECM proteins carefully selected based on the

composition of the in vivo BM. An alternative approach for creating a suitable ECM


27

would be to coculture brain endothelial cells with astrocytes and pericytes, which

secrete many of the specific BM proteins that endothelial cells rely on in vitro.

Figure 6. Schematic of common 2D and 3D monoculture and coculture BBB models. In

2D Transwell cultures, endothelial cells are seeded on a porous membrane hanging

inside a culture well. In coculture Transwell models, other NVU cell types can be added

to the bottom of the membrane and/or to the bottom of the well. In 3D microphysiological

systems (MPS), endothelial cells can be seeded in cylindrical hollow channels. Systems

can contain single or multiple channels separated by porous membranes. In multiple

channel systems, one channel can be used to culture the endothelial cell tube, and an

adjacent tube can be used to culture other NVU cell types.

Characterization of in vitro BBB models

Expression and localization of endothelial cell marker proteins are frequently used to

characterize BBB models. Specifically, the expression of TJ proteins, such as occludin,

claudin-5 and ZO-1, are used to verify that endothelial cells form TJs. TJs between

brain endothelial cells create a physical barrier, which reduces permeability and

Delsing, Louise

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sustains polarization of transport proteins. Furthermore, the tightness of the barrier

needs to be sufficient in order for any measurement of efflux activity to be meaningful.

If the endothelial cells express efflux proteins but do not physically restrict the

paracellular space, which is the case with many primary cell models, it is difficult to

verify the activity of the efflux transporters. TEER measurements are the most

common way of assessing how well the cells limit the transport in the paracellular

space. High TEER represents a low paracellular transport and a tight monolayer. TEER

measures the conductance of the cell membranes, a measurement that is relatively

straightforward to perform in 2D Transwell cultures but can be very difficult to

perform in microfluidic 3D models. TEER measurements are not feasible in vivo but

in vivo TEER values have been estimated to be above 1000Ohm x cm2. Even though

systems with physiological TEER will have negligible paracellular transport, the

permeability across a monolayer is not linearly correlated with the TEER. In addition,

TEER is affected by other parameters such as the composition of solutes in the

medium, temperature and handling of the equipment during the measurement. Hence,

permeability measurements using small molecular tracers should be performed in

addition to TEER measurements. Fluorescent tracers provide an opportunity to follow

permeability in real time and assessment of permeability with a fast and easy plate

reader analysis. Small fluorescent substances are commonly used for such analyses,

for example, fluorescent dextrans of different sizes, sodium fluorescein and Lucifer

yellow.

Evaluations of expression and functionality of efflux transporters are important to

understand the translatability of BBB models when determining entry of novel

therapeutics into the CNS. The two most studied transporters are P-gp and BCRP. In

2D Transwell systems where bi-directional permeability measurements are possible,

efflux ratio is often used to determine if a substance is affected by efflux transporter

activity. As efflux transporter location is polarized and mostly located in the apical

(blood facing) membrane the transport of an efflux transporter substrate is lower in the

apical to basolateral direction (blood to brain) than in the basolateral to the apical (brain

to blood) direction. The efflux ratio is the ratio between the apical to basolateral and

the basolateral to the apical transport. Consequently, the efflux ratio of a substance

with equal permeability in both directions not affected by efflux is 1, while substances

affected by efflux typically has efflux ratios of 2-10. Generally, a substance with efflux

ratio greater than 2 is considered an efflux transporter substrate. Investigation of efflux

transporter functionality can also rely on analysing the permeability of known efflux


29

transporter substrates with and without inhibition of the efflux transporters. For

example, rhodamine 123 is a known substrate of P-gp and its permeability can be

monitored with or without the addition of the P-gp inhibitor verapamil. In 3D dynamic

systems bi-directional transport measurements are often difficult to perform, hence the

investigation of efflux functionality usually relies on permeability studies of known

substrates with and without inhibition of the efflux transporters.

Astrocyte cocultures have shown to improve barrier properties in many in vitro BBB

models (93, 96, 116). These models use non-contact cocultures, suggesting that the

astrocyte contribution occurs mainly via soluble factors. However, in vivo, astrocytes

have a significant structural contribution to the barrier with astrocytic end-feet

wrapping around the endothelial cells. Due to the difficulties in recreating the

astrocyte-endothelial cell structural connections in vitro, the significance of the

physical interaction of these two cell types remains elusive. Pericyte cocultures have

similarly been reported to be beneficial to BBB models (98, 99, 117), and many of

these have the pericytes in close proximity to the endothelial cells. Pericytes surround

endothelial cells and help regulate capillary diameter and blood flow, which suggest

that the contact between pericytes and endothelial cells is important. In addition, both

pericytes and astrocytes contribute structural components of the BBB by secreting

proteins that constitute the basement membrane.

In summary, for improved translatability to the in vivo situation, a BBB model should

adopt a 3D tubular morphology and the endothelial cells should be subjected to shear

forces. Endothelial cells should express TJ markers and efflux proteins. The

permeability across the endothelial cell layer should be low. BBB model can benefit

from coculture with astrocytes and pericytes, however the intended use for the model

ought to dictate what level of complexity is needed.

iPSC-derived blood-brain barrier models

A model with iPSC-derived cell types could overcome the challenges with

reproducibility and availability of human cells and would provide the possibility for

an isogenic model with all cell types originating from the same human individual. In

addition, using iPSC-derived cells would reduce the need for animals and animal

derived tissues to be used. The establishment of an iPSC-derived BBB model requires

robust and reliable differentiation protocols for derivation of several cell types of the

CNS. As described above, differentiation protocols for endothelial cells, astrocytes and

Delsing, Louise

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more recently pericytes are available. Hence an iPSC-derived BBB model is feasible,

and during recent years, there has been a rapid increase in iPSC-derived BBB models.

The establishment of the brain endothelial cell differentiation protocol by the Shusta

lab in 2012 (87) served as an accelerator for iPSC-derived BBB model work. Most of

the published iPSC-derived BBB models used variations of that differentiation

protocol. Several of these iPSC-derived BBB models have rapidly developed into tools

for investigation of drug permeability studies (87, 116, 118-120), disease modelling

(7, 99, 121-123) and modelling of BBB disruption (124-127). Both monoculture

models of the BBB using only iPSC-derived endothelial cells and coculture models

with endothelial cells and other cell types of the NVU have been established. Coculture

models contained endothelial cells and different combinations of astrocytes, pericytes,

neurons and neural stem cells (93, 98, 116, 128). iPSC-derived coculture models of the

BBB formed monolayers with highly restricted permeability in the paracellular space.

TEER values for coculture models have been reported to be higher than 6000Ohm x

cm2 (128), however the variability in maximum TEER reported between iPSC-derived

models is quite high and others have reported TEER values of ~1000-4000Ohm x cm2

(91, 93, 116). Permeability of passively diffused soluble substances such as fluorescein

have been reported to be in the range of ~1-5 x 10-7cm/s in iPSC-derived BBB models

(90, 93). This was substantially lower than the permeability achieved in brain

endothelial cell line cultures of ~12-15 x 10-6cm/s (129) and can be compared to in

vivo measurements in rat of 2.7 x 10-6cm/s (130). Efflux by P-gp is commonly

investigated and found to be active in iPSC-derived BBB models, however, the activity

of P-gp was not affected by coculture (93, 116, 128). Across iPSC-derived BBB

models coculture with astrocytes appears to increase the barrier restriction capacity of

the endothelial cells. Results from pericyte cocultures are more conflicting, with some

studies showing improved barrier restriction with coculture (88, 90, 98, 128) and other

reporting no differences (116, 117). Interestingly, these conflicting results have been

reported for both cocultures with iPSC-derived pericytes and primary pericytes. One

of these studies demonstrated that even though pericyte coculture had no effect on

endothelial cells under normal conditions, pericyte coculture had the ability to rescue

barrier properties in stressed endothelial cells and allowed endothelial cells to maintain

high TEER over longer culture time. This suggested that the pericyte contribution in

in vitro BBB models was maintenance rather than induction (117). These discrepancies

highlight the numerous factors contributing to variability in complex multicellular

models, such as the iPSC-line background, culture conditions and assay conditions.

Even though some differentiation protocols have proven to be highly robust and


31

transferrable between different labs and applications, variability is an issue when

comparing models. This was exemplified in a study comparing the differentiation

capacity of four different iPSC lines to isogenic BBB models including endothelial

cells and astrocytes (131). Even though many iPSC-derived BBB models have been

developed, characterized and used in different applications, several questions remain.

To facilitate the use of iPSC-derived BBB models in disease modelling and drug

discovery, the mechanism of BBB induction by coculturing cell types and the

expression and functionality of more brain-specific transporters need to be thoroughly

investigated. Furthermore, additional studies of drug permeability prediction are

needed.

Microfluidic models

Recently several microfluidic BBB models containing iPSC-derived endothelial cells

have been reported (95, 117, 132-136). These models aim to recreate the 3D

morphology of vessels and allow the cells to interact under more physiological

conditions. In these models, the cells are subject to shear forces introduced by flow,

similar to the in vivo conditions in brain blood vessels. The shear forces that affect the

cells are determined by the vessel diameter, the viscosity of the flowing liquid and the

flow rate. Human micro-vessels and shear stress have been studied in the eye where

diameters ranged between 6 and 24µm. The shear stress was measured to be between

2.8 and 95dyne/cm2, the calculated average shear stress was 15.4dyne/cm2 (137).

These findings can be compared to measurements of vessel diameters in the human

motor cortex where the perforating capillaries have a diameter ranging from 5 – 8µm,

the arterioles have a diameter ranging from 10 – 15µm and the venules have a diameter

ranging from 16 – 20µm (138). Brain post capillary venules are characterized by

diameters of around 100μm, a relatively thick basement membrane, and a wall shear

stress of 1–4dyne/cm2 (114, 138). Compared to the human in vivo brain vasculature,

most BBB microphysiological systems (MPS) recreate an environment, which is

similar to the vessel diameter and shear forces of post capillary venules. Similarly to

the static models, the majority of iPSC-derived MPS models have used variants of the

protocol proposed by Lippmann et al. (88) to derive brain endothelial cells and cultured

them as monocultures or cocultures. Several coculture models used primary sources

for pericytes and astrocytes (95, 132, 134). However, recently two fully iPSC-derived

models have been reported, one using iPSC-derived pericytes (117) and one using

iPSC-derived neural cells for coculture (133). iPSC-derived BBB models in MPS

Delsing, Louise

32

formed tight barriers and showed low permeability, below 5x10-7cm/s for 10kDa

dextran in both monoculture of brain endothelial cells (135, 139) and coculture models

(95, 132, 133). Similarly to static 2D models, MPS BBB models showed active P-gp

efflux (132, 135, 139) and improvements in barrier phenotype after coculture (133,

134).

Comparing 2D and 3D models to elucidate effects of more physiological culture

conditions and addition of shear stress is challenging. Consequently, effects of

introducing shear stress on iPSC-derived brain endothelial cells are not well studied.

Evaluation of permeability between static and dynamic models will have inherent

differences in physical prerequisites. Furthermore, elucidating what differences flow

creates is difficult because medium volumes in many MPS are low and therefore flow

is necessary to supply the cells with oxygen and nutrients. Hence, creating a static

culture in these systems is often not possible and direct comparisons between static

and dynamic conditions are not feasible. Despite these difficulties, comparisons of

MPS cultures with and without shear stress have been reported (136, 139). It was

concluded that introducing shear stress on iPSC-derived endothelial cells had other

effects than introducing shear stress on primary endothelial cells. iPSC-derived

endothelial cells subjected to shear stress had lower apoptosis, lower proliferation and

lower cell mobility, however no change in TJ proteins were found, even though shear

stress served to increase contact area between cells (136). Another recent study

comparing iPSC-derived brain endothelial cells under shear stress and static conditions

showed that iPSC-derived brain endothelial cells that were subject to flow had lower

passive paracellular permeability but no difference in efflux transporter activity (139).

However, expression levels of several tight junction and endothelial cell markers have

been found to depend on the flow rate in a fully iPSC-derived model (133). Studies of

primary brain endothelial cells revealed interesting effects of shear stress in culture.

Under flow, these cells went from a mostly anaerobic metabolism producing lactate to

a mixed aerobe and anaerobe metabolism producing both lactate, H2O and CO2 (115).

There have been speculations that, as the BBB tightens more active and energy

demanding transport is necessary and hence the endothelial cells make use of a more

aerobic metabolism, which is more efficient in generating ATP. Another speculation

was that the metabolism is dependent on blood flow and thus oxygen levels. When the

blood flow is low and hence the oxygen availability is low, a more aerobic metabolism

can be utilized.


33

In addition, MPS models have been suggested to be more compatible with permeability

screening as continuous sampling is possible in dynamic systems. However, many

MPS models require complex laboratory setups that are incompatible with high-

throughput screening. There is evidence that MPS may be useful for providing a

physiologically more relevant culture environment for iPSC-derived BBB models.

However, many questions remain regarding how adding flow and a 3D culture

environment benefit iPSC-derived BBB models.

Delsing, Louise

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35

5 AIMS

The thesis aims were to develop human iPSC-derived models of the BBB that display

barrier phenotype and characterize these models in terms of brain endothelium-specific

properties. The model development was based on investigations of mechanisms

important for barrier formation in iPSC-derived endothelium and development of high-

quality cells to use in the model. The possibilities to use the model in drug discovery,

and in determination of brain-penetrating capacity of drugs were specifically

considered.

The specific aims were:

• To increase the knowledge of the molecular mechanisms behind the

restricted permeability across iPSC-derived endothelial cells and to identify

transcriptional changes that occur in iPSC-derived endothelial cells upon

coculture with relevant cell types of the neurovascular unit. (Paper I)

• To develop and identify high-quality astrocytic cells, and to evaluate the

biological relevance and model diversity between astrocytic models. (Paper

II)

• To evaluate a xeno-free differentiation process of iPSCs to astrocytes, and

subsequently evaluate the capacity of xeno-free-derived astrocytes to induce

BBB properties in iPSC-derived endothelial cells. (Paper III)

• To generate a biologically more relevant iPSC-derived BBB model and to

improve compatibility with high-throughput substance permeability

screening by developing a microphysiological dynamic model. (Paper IV)

Delsing, Louise

36


37

6 METHODOLOGICAL CONSIDERATIONS

The following section describes the reasoning behind method choices and discusses

advantages and limitations of the methods used in this thesis. Detailed methods for

specific experiments can be found in Paper I-IV.

Ethics

Human iPSC lines C9 and C1 used in these studies were derived with written informed

consent by the donors or their parents. Generation of iPSCs were reviewed and

supported by regional ethical consent boards. R-iPSC1j were generated from BJ

fibroblast (CRL-2522) purchased from ATCC, Manassas, Virginia, USA, in

compliance with the ATCC materials transfer agreement. AF22 were generated

from primary fibroblast purchased from Cell Applications Inc., San Diego, CA,

USA and were used in compliance with vendor agreements. ChiPSC22 iPSC line

were purchased from Takara Bio, Kusatsu, Japan and used in compliance with

vendor agreements. SFC-SB-AD2-01 iPSC line were obtained through the Innovative

Medicines Initiative Joint Undertaking StemBancc and used in compliance with user

agreements. Primary brain endothelial cells (Cell Systems, Kirklans WA, USA),

astrocytoma cell line CCF-STTG1 (ATCC, Manassas, VA, USA), human

embryonic kidney cell line HEK293 (ATCC, Manassas, VA, USA), human brain

astrocytes HBA (Neuromics, Edina, MN, USA), immortalized brain endothelial cell

line CMEC/D3 (Merck, Kenilworth, NJ, USA), and iPSC-derived iCell Astrocytes

(FUJIFILM Cellular Dynamics, Inc, Madison WI, USA) used are commercially

available and used in compliance with vendor agreements.

Cells

Even though iPSC lines generally are of high quality and exhibit the gold standard

requirements for stem cells, there are inherent differences between cell lines. These

differences have previously been investigated in isogenic BBB models and subtle

variances in marker expression and maturation state were observed (131). Observed

differences may depend on multiple factors, such as genetic makeup of the donor, the

cell type and method that was used to derive the iPSCs, and under what culture

conditions the iPSCs have been maintained. Due to the inherent differences between

lines they may respond differently to differentiation, coculture or other treatments.

Delsing, Louise

38

Thus, it is important to include several iPSC lines in the experiments to be able to draw

general conclusions. In this work, the aim has been to include 2-3 lines per experiment.

In some cases, one line has been used in parts of the analyses after sufficient

verification that similar outcome for other lines is to be expected.

Differentiation of iPSC-derived endothelial cells

Differentiation of iPSC-derived endothelial cells was evaluated using one protocol

generating brain-specific endothelial cells through a differentiation process that

includes spontaneous neuroectodermal coculture (88, 90) and one general endothelial

cell differentiation protocol (97), in which cells are generated via mesenchymal

induction. Most of the work was performed using cells derived with the brain-specific

endothelial cell differentiation protocol. This protocol was first published in 2012 (87),

modifications and improvements of the protocol have since then been implemented

(88, 90). Until very recently this protocol was the only published brain endothelial cell

differentiation protocol generating endothelial cells capable of forming tight

monolayers. It has proven to be robust and has been successfully implemented with

many different iPSC lines and labs for numerous applications. iPSC-derived

endothelial cell characterization is based on both expression of endothelial cell markers

and ability to form a restrictive barrier, further discussed below.

Differentiation and functional characterization of iPSC-derived astrocytes

Differentiation of iPSC-derived astrocytes was performed using a modified version of

a previously published protocol (103), which provides the possibility to generate

astrocytes from neural progenitors within a month. Astrocytes are a very diverse cell

type, and as such, finding a common marker to identify astrocytes has proven difficult.

GFAP is commonly used as an astrocytic marker, but not all astrocytes in the human

brain express GFAP (140), and GFAP expression is commonly recognized as a marker

for reactive astrocytes (29). Preferably, expression of several astrocyte associated

proteins, in combination with functional testing, should be used to characterize

astrocytes. In these studies, glutamate uptake, inflammatory response and calcium

signalling were used to evaluate important functionality of astrocytes. Glutamate

uptake analysis was performed by measuring the decrease of glutamate in the culture

over time using a colorimetric assay, with or without the addition of glutamate


39

transporter inhibitors. To avoid interference with the glutamate detection process, non-

substrate inhibitors were used. All glutamate uptake measurements were normalized

to number of cells in each well through either nucleus counting or double stranded

DNA content. Astrocytes are active in the immune response of the CNS and

inflammatory response assays were performed by measuring secretion of cytokines

indicating reactive activation with or without stimulation with the proinflammatory

factors TNFα and IL-1β, known to be produced by microglia, leukocytes and

astrocytes. Calcium signalling analysis was performed using a neutral calcium

indicator that can cross the cell membrane. Once inside the cell, the indicator is cleaved

by esterases activating its calcium-binding properties and rendering it charged, which

prevents transport out of the cells. When calcium is released, it is bound by the

indicator and increases its fluorescence. Calcium signalling was measured in response

to ATP and glutamate and was compared to an injection control as changes in shear

stress has been shown to influence calcium signalling.

Characterization of protein and mRNA expression

Protein expression was analysed using immunocytochemistry (ICC) for intracellular

proteins and enzyme-linked immunosorbent assay (ELISA) for secreted proteins. ICC

provides the possibility to visualize the location and pattern of expression of the

investigated protein that other methods, such as western blot, do not provide. When

studying important structural components such as TJs it is helpful to be able to assess

if TJ proteins locate to the cell junctions and if there are continuous TJs between cells,

which is a requirement for permeability restriction across the cells. Expression analysis

through qPCR is a useful tool to detect changes in expression of certain genes of

interest. A change in mRNA may indicate that there are changes in protein expression,

even if the relationship between mRNA and protein expression is not perfectly

correlated. Equally important, a protein can be present in a cell without exhibiting its

function. Hence, the characterization of cells should contain analysis on mRNA level,

protein level and functional analysis. Complementary mRNA and ICC analyses are

highly desirable, but not always feasible when quality antibodies to detect the protein

of interest are not available. Developing specific antibodies against membrane proteins

has been a long-standing challenge due to the difficulties in delivering enough protein

in pure enough forms with intact tertiary structure to immunize. This can be a particular

challenge when studying the BBB where many of the proteins and transporters of

Delsing, Louise

40

interest are membrane-bound. qPCR analysis also comes with the need to carefully

consider methods for normalization of samples. For experiments in this thesis three

housekeeping genes were tested and the most stable one was selected using the

NormFinder algorithm (141). Adding shear stress to a BBB model system has been

shown to cause major shifts in fundamental processes such as metabolism and protein

production (115), because of this five housekeeping genes were evaluated with

NormFinder for experiments in Paper IV.

Barrier integrity assays

Barrier integrity assays are performed using TEER and permeability analyses of

fluorescent tool compounds and drug substances. TEER measures the conductance of

the cell membranes: high TEER represents a low paracellular transport and a tight

monolayer. TEER is a fast, easy and effective method of assessing tightness of a cell

monolayer. However, it is important not to rely solely on TEER for permeability

measurements as this method is quite variable and exact values can be problematic to

compare between labs. A universal permeability measurement such as apparent

permeability (Papp) is preferable when comparing models and hence both of these

methods can be used together to get comprehensive information on permeability across

a BBB model. Efflux transporters are an important aspect of permeability restriction

across the BBB. In this work, efflux transporter analyses have focused on the efflux

transporters P-gp and BCRP as they are both highly expressed in the BBB and have

been found to limit the brain permeability of many drug substances. Analysis of efflux

protein activity and polarization was performed through directional permeability

analyses and permeability studies with and without inhibiting the efflux transporters.

Fluorescence-based assays where the permeability of a fluorescent substrate is

monitored provides significantly simpler analyses compared with other methods of

determining substrate concentrations such as mass spectrometry or radiolabelling of

substances. Even though these assays are generally very useful they have certain

limitations. For example, any assay using a P-gp substrate to detect the interaction with

P-gp of another substance will be limited by the affinity of the substrate to P-gp. It will

be difficult to detect effects of substances with a lower affinity to P-gp than the chosen

substrate. In addition, non-substrate inhibitors will be difficult to separate from

substrates with high affinity having prolonged attachment to P-gp, which slows down

its activity rate.


41

RNA sequencing and pathway analysis

RNA sequencing (RNAseq) is a powerful method for identifying and characterizing

cell cultures by investigating the expression of all genes under certain conditions. It

allows for analysis of the similarity and differences between samples and of larger

groups of genes related to a specific functionality or a specific signalling pathway. An

important limitation of the technique is that it gives cross-sectional data and does not

capture dynamic changes. Signalling pathways regulated by genes identified as

differentially expressed by RNAseq can be detected with pathway enrichment analysis.

Such pathway enrichment analyses depend on lists of genes annotated in databases to

belong to certain pathways. Overrepresentation of the members of such gene lists

among the differentially expressed genes is then investigated. Even though signalling

is a dynamic process, evaluating expression of most of the components of a signalling

pathway can provide insight into differences between samples. However, experimental

verification is still required. RNAseq experiments in this thesis examines bulk RNA,

the collected RNA from a pool of cells. Another RNAseq method is single cell

RNAseq, where data is collected for each cell separately. This kind of analysis would

be interesting to perform in future analysis of iPSC-derived BBB model as it can

provide further insight about heterogeneity within the cell population. However, it

requires a more complex and time-consuming analysis compared to bulk RNAseq.

Microphysiological culture systems

To create a more physiological culture setting for brain endothelial cells efforts are

under way to adapt cultures to MPS. Most MPS use tubing and pumps to create a

dynamic flow culture, which provides shear stress and flow that are unidirectional and

adjustable. However, these systems require complicated laboratory setups that are very

difficult to run in high-throughput. The Organoplate used in this thesis provides 40

units within one 384 well plate creating a system which is suitable for automated high-

throughput screening, but has a bi-directional flow driven by gravity on a tilting

platform. Thus, the Organoplate system provides a compromise in which a

unidirectional flow system is sacrificed in favour of a system with bi-directional flow

suitable for high-throughput screening. However, rat brain capillary endothelial cells

in vivo experience flow fluctuations, extended stalls and even reversals of direction

under physiological conditions (142). Even though the Organoplate clearly has the

potential to be used in high-throughput screening, this has not yet been reported for

any BBB models. The Organoplates’ parallel membrane free channels are suitable for

Delsing, Louise

42

barrier integrity assays relevant specifically to the BBB, vasculature and renal models,

which represent the majority of the developed models in this system. The Organoplate

endothelial tube compartment is 400 x 220µm, when plates are perfused at a 7° angle

the cells are subject to a shear stress of 1.2dyne/cm2, which is comparable to the shear

stress in post capillary venule in the brain. Consequently, the Organoplate system is a

feasible option for creating a perfused, 3D, high-throughput compatible iPSC-derived

BBB model.

Statistical analysis

Most of the statistical tests in this thesis were performed using an ANOVA or double-

sided Student’s t-test. Student’s t-tests are used to compare means of two groups e.g.

coculture vs. monoculture, L2020 differentiation vs. LN521 differentiation or

Transwell vs. MPS. In this work such comparisons were only made within iPSC lines.

To perform multiple comparisons ANOVA was used and p-values were corrected for

multiple comparisons using Tukey’s or Dunnett’s methods if all groups were compared

to each other or if treatments were compared to a control, respectively.

Experiments in this thesis generally rely on three independent replicates and three

technical replicates for each analysis. Technical replicates are included to control for

variability in the testing protocol. Cell culture data brings controversy with regards to

what is considered a biological replicate and what is simply a technical replicate. In

iPSC-derived models in this thesis, replicates represent different batches of

differentiation. However, one could argue that since these cells all come from the same

parent iPSC line using three differentiation batches are only technical replicates of the

differentiation protocol. Hence multiple iPSC lines have been used as each iPSC line

can be viewed as a biological replicate. The statistical comparisons were typically

made within the same line and subsequent conclusions were made based on what

trends were observed for all lines. Similar issues can be raised for immortalized cell

lines, whether a new batch of frozen cells is a biological replicate or if a new isolation

is needed to obtain a new biological replicate. For cell lines, such as the CMEC/D3

used in this thesis, replicates come from individual cultures prepared separately but

originating from the same stock. One possible solution to this problem would be to

include several different brain endothelial cell lines. In this thesis, the brain endothelial

cell line was used as a reference and was not the primary the focus of the work, hence

only one line was included.


43

RNAseq data is not normally distributed; it has a negative binomial distribution.

Transcript abundance is estimated by read counting, so the measured variable is

discrete. In addition, RNAseq data is heteroscedastic, meaning that variance in

expression depends on the mean. All of these features have to be considered in

visualization and statistical testing of the data. RNAseq data was analysed using R and

the DESeq2 package. To perform visualizations such as a principal component analysis

(PCA), the data was normalized using the regularized log2 method to correct for

heteroscedasticity and sequencing depth. Statistical analysis was performed with a

generalized linear model and the Wald test was used for significance testing.

Corrections for multiple comparisons were done using the Benjamini-Hochberg

method. The threshold for false discovery rate was set to 5%. For visualization in bar

plots, the data was normalized for sequencing depth and gene length using the

fragments per kilobase million (FPKM) method for paired-end sequencing. Pathway

analysis was performed with the DAVID tool using Fisher’s exact test to investigate if

any pathways were enriched among the differentially expressed genes. The Benjamini-

Hochberg correction for multiple comparisons were used

Delsing, Louise

44


45

7 SUMMARY OF FINDINGS

Paper I: Barrier Properties and Transcriptome Expression in Human iPSC-Derived Models of the Blood-Brain Barrier

There is a need for better in vitro models of the BBB as immortalized cell lines and

primary brain endothelial cells have limited capacity to recreate barrier restriction

when cultured in vitro. iPSC-derived brain endothelial cells have been shown to exhibit

high barrier function in vitro. This work was designed to set up a fully iPSC-derived

model of the BBB containing endothelial cells, pericytes, astrocytes and neurons that

recapitulate barrier functions of the BBB in vitro. It investigated how different methods

of deriving endothelial cells from iPSC affected their ability to serve as BBB models.

Furthermore, the BBB specification of endothelial cells in coculture with other cell

types of the NVU, was investigated. Both the functional changes in barrier restriction

and the transcriptional changes induced by coculture were evaluated. Two published

protocols for generation of endothelial cells were compared, one which generates an

unspecified subtype of endothelial cells and another which generates brain-specific

endothelial cells. The endothelial cells in monoculture and coculture with iPSC-

derived pericytes, astrocytes and neurons were then compared. The results showed that

the brain endothelial cell-specific protocol generated a BBB model with highly

selective permeability. These cells had markedly improved barrier properties

compared with the cells derived using the other protocol, which generated unspecified

endothelial cells. The brain-specific endothelial cells had high TEER and low passive

permeability of fluorescein. Coculture of brain specific endothelial cells with iPSC-

derived astrocytes, pericytes and neurons improved barrier properties and the

cocultured brain endothelial cells showed higher TEER and lower fluorescein

permeability compared to the monocultured brain endothelial cells. Efflux

transporters, such as P-gp and BCRP regulate the brain penetration of many drug

molecules and the activity of these efflux transporters are very important for correct

modelling of permeability, especially of drug-like substances, which often are small

and able to diffuse across cell membranes. The brain-specific endothelial cells in

monoculture and coculture showed active efflux by P-gp, while only the cocultured

endothelial cells showed active efflux by BCRP. No activity of P-gp or BCRP, could

be detected in the nonspecific endothelial cells in either monoculture or coculture. The

Delsing, Louise

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apparent permeabilities of six drug substances were tested and the brain specific

coculture model could distinguish between CNS permeable and non-permeable

substances. To understand molecular mechanisms behind the improvement seen in the

coculture, the transcriptome of iPSC-derived endothelial cells in monoculture and

coculture were compared. Coculture increased the expression of both junction-

associated mRNAs and brain specific transporter mRNAs. A pathway analysis

revealed enrichment of changed genes in the WNT, TNF and Pi3K-Akt pathways.

These data suggested that differentiation towards brain-specific endothelial cells is

needed to obtain endothelial cells with the capacity to form a tight barrier in vitro.

Furthermore, non-specific endothelial cells derived from iPSCs did not develop brain-

specific endothelial cell properties by coculture with NVU cell types. Our results

suggested that the coculturing cell types exerted an influence on the brain endothelial

cells through the WNT, TNF and Pi3K-Akt pathways, ultimately leading to a more

highly restricted barrier in coculture. This work highlighted the plasticity of the iPSC-

derived brain endothelial cells and the ability of coculture with other cell types of the

NVU to enhance their barrier phenotype.

Paper II: Human iPS-Derived Astroglia from a Stable Neural Precursor State Show Improved Functionality Compared with Conventional Astrocytic Models

This work was aimed at developing iPSC-derived astrocytes and to evaluate their

usefulness as an astrocyte model compared to conventional astrocytic models. Models

were compared in terms of transcriptome, protein expression and functionality.

Astrocytes were derived from lt-NES, a homogeneous and stable neural precursor,

which provides shorter differentiation time compared to starting from iPSCs. In this

work it was established that lt-NES can acquire gliogenic potency by expression of

key gliogenic transcription factors SOX9 and NFIA. Astrocytes derived from NES

(NES-astro) expressed many key glia marker proteins such as brain lipid-binding

protein (BLBP), S100B, CD44, SLC1A3 and several astrocyte related mRNAs such

as GFAP, aldehyde dehydrogenase 1 family member L1 (ALDH1L1) and aquaporin 4

(AQP4). NES-astro were compared to primary human astrocytes (phaAstro), the

astroglioma cell line CCF-STTG1 (CCF) and the commercially available iPSC-derived

astrocytes (iCellAstro). In addition, the neural stem cell lt-NES and human embryonic

kidney cell line (HEK) served as neural and non-neural controls respectively.


47

Transcription and protein expression analysis revealed large differences between the

models. As there is no reliable marker or transcription identity that fully specifies

astrocyte biology, several important functional properties were compared between the

different models. Removal of excess glutamate in the synaptic cleft is a critical

astrocytic function that ensures reliable synaptic signalling and prevents excitotoxicity

in neurons. The glutamate uptake mainly occurs through the sodium dependent

glutamate uptake transporters EAAT1 (SLC1A3) and EAAT2 (SLC1A2). NES-astro

showed active glutamate uptake through SLC1A3 that was not observed in any of the

other models. Another important function of astrocytes in vivo is to produce an

inflammatory response. This capacity was assessed by stimulation with inflammatory

cytokines and subsequent evaluation of the response by measuring secreted IL6 and

IL8. Dose dependent inflammatory responses were detected in NES-Astro, these

responses were significantly different from the response in NES. Inflammatory

responses were detected in the other models as well, however the baseline IL6 and IL8

levels were high in phaAstro, CCF and iCellAstro while no base line secretion was

detected from NES-astro. This suggests a completely inactive inflammatory state of

NES-astro at baseline. Calcium responses to ATP and glutamate were evaluated and

both phaAstro and NES-astro showed response to ATP. Only NES-astro showed

calcium response to glutamate. Next, the ability of the models to serve as screening

platforms for apoE secretion were evaluated. Cholesterol and lipid metabolism in the

brain is regulated by astrocytes, and the lipoprotein transporter protein apoE is

predominantly produced by astrocytes. Given the strong genetic link between apoE

isoform and Alzheimer’s disease, apoE is a highly interesting target in drug

development. As such, a high-throughput compatible pilot screen was set up to

evaluate apoE secretion after treatment with known apoE inducers. Results showed

that none of the substances tested induced apoE secretion in all models, highlighting

that hit-finding depends on the cellular model used. For example, liver X receptor

(LXR) agonists are well documented apoE enhancers that produced very high

responses in CCF but did not produce uniform increased apoE secretion in NES-astro

or phaAstro. Interestingly, substances acting on the cholesterol biosynthesis pathway

were identified to increase apoE secretion in both phaAstro and NES-astro, while no

effect was seen in CCF. These results suggested that caution needs to be taken when

choosing an astrocyte in vitro cell model for screening, especially if primary and

secondary screens are undertaken with different cell types. In summary, NES-astro

showed high similarity to phaAstro, demonstrated several functional characteristics

Delsing, Louise

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and astrocytic markers and represented an astrocytic model with high biological

relevance.

Paper III: Enhanced Xeno-Free Differentiation of hiPSC-Derived Astrocytes Applied in a Blood-Brain Barrier Model

To improve astrocyte differentiation and adapt astrocyte differentiation to xeno-free

conditions, a comparative study was made between differentiating astrocytes on

murine sarcoma derived laminin (L2020) and human recombinant laminin 521

(LN521). We showed in Paper I that iPSC-derived BBB models could benefit from

coculture with iPSC-derived astrocytes, pericytes and neurons. In addition, our

comparison in Paper II of astrocyte models shows that NES-astro is a more reliable

astrocyte model than iCellAstro, which was used in the BBB model in Paper I.

Consequently, the BBB model may benefit from coculture with NES-astro rather than

iCellAstro. Furthermore, several published models have achieved improvements in

BBB models using only astrocyte coculture. Our previous data suggest that NES-Astro

can be differentiated on laminin 521 with similar transcriptional profile to L2020-

differentiated astrocytes, but no functional comparison of xeno-free and conventional

NES-astro differentiation was performed. Hence, this work was aimed at comparing

functionality and barrier inducing capacity of astrocytes differentiated from lt-NES on

L2020 and LN521. Laminins are the most abundant component of the BM, which lines

the brain blood vessels. In addition to their structural functions, laminins play essential

roles in the organization of the BM and in the regulation of cell behaviour. Several in

vitro cell models have shown enhanced functional development when cultured on

specific laminins. Astrocytes derived on L2020 and LN521 showed expression of

astrocyte-related proteins and mRNAs such as BLBP, GFAP and S100B. In addition,

astrocytes differentiated on LN521 had higher expression of GFAP, S100B,

ALDH1L1, Ang-1 and GDNF mRNAs. Glutamate uptake analysis showed that both

L2020- and LN521-differentiated astrocytes have functional uptake of glutamate

through EAAT1 (SLC1A3). However, LN521-differentiated astrocytes had higher

expression of EAAT1 on both protein and mRNA levels. The reduction in glutamate

uptake upon inhibition of EAAT1 were greater in two of the biological replicates of

astrocytes differentiated on LN521 compared to astrocytes differentiated on L2020,

this could be an effect of the increased protein and mRNA levels of EAAT1. Further

investigating expression of mRNA involved in the glutamate metabolism revealed that


49

astrocytes differentiated on LN521 had higher expression of the glutamine exporter

SNAT3. Together with the increased levels of EAAT1 this suggested that the

glutamate metabolism was affected by differentiation on LN521. Proteins secreted by

astrocytes have many important functions in the CNS and a large part of the influence

that astrocytes have on brain endothelial cells is via secreted proteins. Astrocytes

differentiated on LN521 secreted more Ang-1 and S100b compared to astrocytes

differentiated on L2020. This is in agreement with increased mRNA levels of Ang-1

and S100B in astrocytes differentiated on LN521. The capacity of the astrocyte to

induce barrier properties in a xeno-free BBB model was subsequently investigated.

iPSC-derived brain endothelial cells were derived using a xeno-free version of the

protocol used in Paper I and cocultured with astrocytes differentiated on LN521 or

L2020. Both astrocytes differentiated on LN521 and L2020 improved the barrier

properties of brain endothelial cells in coculture. No differences were observed in

TEER, but a slightly lower passive permeability was observed in coculture with L2020

differentiated astrocytes. Interestingly, brain endothelial cells cocultured with LN521

astrocytes showed higher expression of VE-cadherin, one of the improvement points

previously identified for the BBB model in Paper I. Furthermore, the increased

expression of ABCB1 mRNA and the decreased expression of caveolin 1 (Cav1) in

brain endothelial cells appeared to depend on coculture with differentiated astrocytes

and was not observed in coculture with NES. This suggested that coculture with more

mature astrocytes is beneficial. In conclusion, these results show that astrocytes can be

derived on LN521 and used in a xeno-free iPSC-derived BBB model in vitro with

similar results as L2020 derived astrocytes. In addition, astrocytes differentiated on

LN521 may display a more mature phenotype with a higher secretion of factors

important for BBB formation in iPSC-derived endothelial cells such as Ang-1.

Paper IV: An iPSC-Derived Microphysiological Blood-Brain Barrier Model for Permeability Screening

In vitro BBB models have been hampered by lack of physiological structural

arrangement of the cells and inability to recreate the physical forces that affect brain

endothelial cells in vivo. To create a more physiological culture setting for brain

endothelial cells this work was aimed at adapting the BBB model to an MPS. In these

systems, the cells will be subject to both a 3D culture environment and dynamic flow

introducing shear stress, both of which have been proven important for functional

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development of the neurovasculature. Furthermore, to address the need to develop

BBB models that are compatible with high-throughput screening, the MPS used in this

study is well-suited for high-throughput applications. An MPS BBB model was created

by culturing iPSC-derived brain endothelial cells in the Organoplate MPS, which has

the same format as a regular 384 well culture plate and contains 40 microfluidic units

per plate. The flow through the Organoplate was bidirectional and gravity-driven.

Culture conditions of iPSC-derived brain endothelial cells in the Organoplate were

optimized and BBB-specific marker expression and barrier integrity were analysed.

The data was compared with data obtained from cultures in the commonly used

Transwell system (used in Paper I). The iPSC-derived brain endothelial cells showed

expression of the brain endothelial cell marker proteins ZO-1, Glut-1, occludin and

claudin-5, and formed leak-tight tube structures in the MPS, which had negligible

permeability to 4.4kDa dextran and very low permeability to the P-gp substrate

rhodamine 123. Both the 3D MPS and the static 2D Transwell cultures had low

permeability of 4.4kDa dextran and rhodamine 123. Comparison of mRNA expression

of brain endothelial cell-associated markers revealed that mRNA levels of VE-

cadherin, CD31 and BCRP were increase in the MPS culture compared with the

Transwell culture. Intracellular accumulation of the BCRP substrate Hoechst was

inhibited by the BCRP inhibitor ko143 in the MPS model but not in the Transwell

model, suggesting that BCRP activity was dependent on the more physiological culture

environment of the MPS. To facilitate high-throughput screening, cryopreservation

and direct seeding of frozen iPSC-derived brain endothelial cells into the MPS were

evaluated. Brain endothelial cells were cryopreserved and seeded directly into the

Organoplate without effects on barrier integrity or mRNA marker expression. Finally,

a pilot screen to identity substances that interact with the efflux transporters P-gp and

BCRP was performed. Known P-gp and BCRP substrates and inhibitors were

evaluated in a fluorescence-based assay where the changes in permeability of the

known P-gp substrate, rhodamine 123, and the known BCRP substrate Hoechst were

evaluated. The MPS BBB model was able to detect all the P-gp and BCRP inhibitors.

In addition, it was able to detect two out of three BCRP substrates. Taken together the

results from this study showed that it was possible to generate a 3D microphysiological

iPSC-derived BBB model with barrier function similar to Transwell models. Even

though the permeability was generally higher in the MPS model the permeability

values were very low for both models. Several brain endothelial cell specific functions

were enhanced in the MPS model compared with the Transwell model. mRNA

expression of junction-associated proteins and BCRP were increased. Interestingly,


51

functional BCRP activity was observed in the MPS but not in the Transwell, and

functional P-gp activity was observed in both MPS and Transwell. Furthermore, thanks

to the 384wp format of the Organoplate and because it was possible to cryopreserve

the iPSC-derived brain endothelial cells in an assay-ready state, this MPS model was

found compatible with high-throughput screening. Consequently, this

microphysiological model of the BBB provides a promising starting point for using

iPSC-derived MPS for predicting brain permeability of novel therapeutics.

Delsing, Louise

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53

8 DISCUSSION

iPSC-derived blood-brain barrier models

This thesis follows the trajectory of the BBB modelling field in general. Static

Transwell models are being replaced by more complex 3D models. There are multiple

reasons why Transwell cultures are replaced by microfluidic systems. MPS provides a

more physiologically relevant culture in several aspects, a three-dimensional spatial

organization of the cells allows for the endothelial cells to form tubes and the

coculturing cells to interact physically with the basolateral face of the endothelial tube.

The importance of shear stress in BBB and vasculature development has been

emphasized in several studies (115, 133, 136) and MPS allows for the addition of flow

and shear stress. Moreover, there are technical aspects that favour the MPS, for

example, the number of cells needed to create an MPS model is substantially lower

and continuous live cell imaging is greatly facilitated in some MPS models compared

with the Transwell models. However, MPS systems similarly have inherent technical

drawbacks, the setup of MPS is often very complicated and requires special laboratory

equipment, TEER measurements are difficult to perform which means that more

laborious assays are needed to assess paracellular permeability. Finally, the throughput

of MPS is typically very low.

The iPSC technology has allowed human cells to be used to a larger extent within BBB

modelling, overcoming issues with availability and quality of primary cells. Primary

brain endothelial cells and cell lines are not able to create restrictive barriers in vitro

(2, 129), as such primary cell models have not served as the gold standard to which

iPSC-derived models can be compared. Extensive research has been performed to

improve primary BBB models, for example, through coculture (143), overexpression

of microRNAs (144) and chemical stimuli (145) with only small improvements in

barrier restriction potential. Interestingly, iPSC-derived brain endothelial cells are able

to create restrictive barriers with paracellular permeability similar to that seen in vivo.

It is still unclear why the iPSC-derived brain endothelial cells are able to recreate the

phenotype of their primary counterparts in vitro. In Paper I, we performed gene

expression profiling to elucidate underlying mechanisms of endothelial cell ability to

restrict permeability. The analysis showed significant changes in the WNT, AKT-Pi3K

and TNF pathways, and a differential expression of occludin and claudins.

Specifically, claudin-8 and -19 mRNA expression, which were found to be upregulated

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by coculture, are associated with reduced permeability (63). Interestingly, we show in

Paper I that the brain endothelial cell line CMEC/D3 and the iPSC brain endothelial

cells derived with the non-brain-specific protocol have higher mRNA expression of

VE-cadherin, CD31 and claudin-5 compared to iPSC brain endothelial cells derived

with the brain endothelial cell specific protocol. Yet, the non-specific endothelial cells

and the CMEC/D3 had higher paracellular permeability, hence our results were not

able to corroborate the previously suggested notion that claudin-5 expression levels

are a determinant of paracellular permeability (2, 116, 146, 147). When investigating

occludin, differences in protein expression correlating with paracellular permeability

were detected, and occludin expression has previously been suggested as a determinant

of TJ permeability in BBB endothelial cells (2, 68, 69). Furthermore, occludin was

recently suggested to have a potential role in increasing TEER levels (128). Taken

together, our results suggest that occludin plays an important role in the iPSC derived

endothelial cells ability to restrict paracellular permeability.

Of note, in Paper II the baseline secretion of cytokines was found to be substantially

higher in iCellAstro compared to NES-astro. Interestingly, it was recently reported that

addition of inflammatory cytokines reduced ZO-1 expression and increased

permeability in an iPSC-derived BBB model (133). Consequently, it is reasonable to

speculate that the BBB models in Paper I and Paper III are affected accordingly, as

iCellAstro was used in Paper I and NES-astro was used in Paper III, however this was

not directly compared.

Comparison to other iPSC-derived blood-brain barrier models

During recent years, there has been a rapid increase in iPSC-derived BBB models.

iPSC-derived BBB models have developed into tools for investigation of drug

substance permeability studies similar to the one in Paper I (87, 116, 118, 119, 133),

disease modelling (7, 99, 121-123) and modelling of BBB disruption (124-127). BBB

models developed in this thesis are comparable to previously published models, both

in marker expression and barrier restriction. As discussed in Paper I, TEER values

obtained of ~ 1250Ohm x cm2 in the coculture were lower than TEER values reported

for similar models of up to ~2500Ohm x cm2 (116) and ~3000Ohm x cm2 (90, 117)

but higher than some other reported TEER values of ~500Ohm x cm2 (93). The inverse

relationship between TEER value and permeability has been shown to disappear at


55

higher TEER levels in iPSC-derived endothelial cells. Threshold values where a higher

TEER no longer corresponds to a lower permeability were found to be 500Ohm x cm2

for the small molecule sodium and 900Ohm x cm2 for a large molecule IgG (119). A

similar study in an animal-derived BBB in vitro model concluded that a TEER value

of above 500Ohm x cm2 only had marginal effects on mannitol permeability (148).

Small differences in TEER were observed between different iPSC lines and variations

between lines in the same experiment are expected, likely due to different donors and

methods of deriving the iPSCs. The TEER measurement is an important tool for

assessing paracellular tightness and restricted permeability in the paracellular space is

of the highest importance for accurate modelling of any transport across the BBB.

However, above approximately 1000Ohm x cm2 the actual TEER value is of little

importance. In vivo TEER values are very difficult to measure due to the invasive

nature of the techniques and requirement of putting electrodes on either side of the

BBB without disrupting it. Despite this, TEER values for vertebrates have been

obtained and values across rat and frog brain endothelial cells have been measured in

the range of 1200–1900Ohm x cm2 (15, 149). In summary, TEER values obtained in

iPSC-derived BBB models are similar to the in vivo TEER and exceed the critical

value for paracellular permeability restriction.

Only a handful of studies have investigated the permeability of drug substances across

iPSC-derived BBB models, but many different substances have been tested in

permeability studies. Some studies display a significant distinction between CNS

permeable and non-CNS permeable substances similar to that in Paper I (116, 118,

119, 133). Substances that have been tested in more than one model include atenolol,

propranolol, caffeine and cimetidine. Atenolol was reported to have a permeability of

approximately 5, 8 and 11 x 10−6cm/s (Paper I, (118, 119)), propranolol

approximately 20 and 40 x 10−6cm/s (Paper I, (119)), caffeine approximately 60 and

100 x 10−6cm/s (116, 119) and cimetidine approximately 1 and 10 x 10−6cm/s (118,

119). In summary, permeability values reported for drug substances are comparable

between published iPSC-derived BBB models. However, there is a need to further

investigate the permeability of drug substances in iPSC-derived BBB models and to

standardize permeability assays to facilitate comparisons between models.

Recently, several iPSC-derived BBB models in MPS have been reported, both

monoculture systems (135, 136, 139) and coculture systems with pericytes and/or

neural cell types (95, 117, 133, 134). In these studies coculture with only pericytes did

not affect permeability (117), but coculture with astrocytes, with or without pericytes

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56

and neurons, resulted in reduced permeability (95, 133, 134) or increased impedance

(132). Permeability measurements using fluorescently labelled dextrans show that

these models have very low permeability, several models reported a permeability of 2-

4 x 10−7cm/s (95) or below detection limit for 10kDa dextrans (117, 135).

Permeability for 4kDa dextran and 3kDa dextran was reported to be in the range of

single digit 10−7cm/s and 1-3 x10−7cm/s. These permeability assessments

correspond well to the ~5 x 10−7cm/s permeability of 4,4kDa observed in Paper IV.

Permeability for 4kDa dextrans across in vivo rat cerebral microvasculature has been

reported to be the 9.2 x 10−7cm/s (130). Additionally, experiments in Paper IV

showed P-gp and BCRP activity in the MPS, similarly, other iPSC-derived BBB MPS

models have reported P-gp activity (132, 133, 135) and BCRP activity (132). A

rhodamine 123 permeability of approximately 0,2-5 x 10−7cm/s was observed for the

MPS in Paper IV, very similar rhodamine123 permeabilities of 1-2 x 10−7cm/s have

been observed in other iPSC-derived BBB MPS (135). Notably, the expression of

several brain endothelial cell markers has previously been shown to depend on flow

(133). Similarly, the expression of several endothelial cell markers was upregulated in

the MPS model compared to the Transwell model in Paper IV, suggesting that this

effect may indeed be flow dependant. Consequently, the MPS model developed in

Paper IV showed very similar permeability properties to other iPSC-derived BBB MPS

and lower permeability compared with in vivo rat data. However, the model may still

benefit from an astrocyte coculture. An important difference between the MPS model

reported in Paper IV and other iPSC-derived MPS BBB models is the compatibility

with high-throughput screening.

Efflux assessment in iPSC-derived blood-brain barrier models

A major requirement for any BBB model to be used in permeability assessments for

safety evaluation of novel drug molecules is the expression and function of the two

main efflux transporters BCRP and P-gp. Evaluation of P-gp and BCRP interactions

are needed for safety studies and are important assessments of drug-drug interactions.

P-gp and BCRP expression and activity were measured in the BBB models in Papers

I, III and IV. Paper I show that both P-gp and BCRP mRNA levels increased after

coculture. No differences in P-gp activity could be detected between monoculture and

coculture, however, BCRP activity was only detected in coculture. Paper III shows that

P-gp mRNA increases when endothelial cells are cocultured with differentiated


57

astrocytes compared to coculture with the neuro epithelial stem cells (lt-NES). No

change was observed in the mRNA expression of BCRP between cocultures with

differentiated astrocytes and NES. Paper IV, examining iPSC-derived endothelial cell

monocultures in MPS and Transwell, showed that BCRP mRNA expression increased

in MPS compared to Transwell and that BCRP activity was detectable in MPS but not

in Transwell. P-gp mRNA expression and activity was not found to be different

between Transwell and MPS. In summary, P-gp is expressed and functional in iPSC-

derived endothelial cells and does not require coculture or culture in MPS for its

activity to be detectable. However, BCRP activity is not detectable in iPSC-derived

endothelial cells in static monoculture. Coculture and MPS culture both increased

BCRP mRNA expression and either coculture or MPS culture is required for detection

of functional BCRP efflux. This is in agreement with studies concluding that iPSC-

derived brain endothelial cells display active efflux by P-gp which is unaffected by

coculture (93, 116, 128). Most published iPSC-derived BBB models do not examine

BCRP activity, but similarly to our results one study reported active BCRP in coculture

(87). Other studies reported both active P-gp and BCRP in monocultures of iPSC-

derived brain endothelial cells. However, these results were obtained using a different

BCRP substrate (131) and after using an adjusted differentiation protocol (91).

Similarly to the results in Paper IV, other iPSC BBB models have shown both active

P-gp (132, 135) and active BCRP (132) in monoculture MPS. However, no direct

comparisons to BCRP activity in 2D culture were made in these studies. In conclusion,

our data suggest that any assay of BCRP-mediated permeability requires a more

complex model with either astrocyte coculture or culture in an MPS.

Blood-brain barrier phenotype of iPSC-derived endothelial cells

Recently, the endothelial identity of iPSC-derived endothelial cells has been

questioned, and claims have been made that these cells are actually neuroepithelium

(150). From the gene expression analysis in Paper I, it was concluded that iPSC-

derived brain endothelial cells may display a mixed endothelial epithelial phenotype.

However, as exemplified in Paper I and IV, the in vitro barrier restriction capacity of

these cells is still highly superior to other brain endothelial cells from immortalized

cell lines, primary cells and iPSC endothelial cells derived with other protocols. In

addition, we showed in Paper I that the expression of the important efflux transporter

BCRP was higher in iPSC-derived brain endothelial cells compared to iPSC

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58

endothelial cells derived with a non-specific protocol. Even though the protein

expression signature of brain endothelial cells derived from iPSCs with the brain

endothelial cell specific protocol may be mixed, these cells display exceptionally high

tightness and expression of BBB specific transporters. As such, they are a very relevant

human model system that can be used for permeability assessments. In vitro cell

models will never be able to recapitulate the full complexity of the in vivo biology and

in the interest of usability, models need to be simplified versions of the modelled

process or structure. As discussed in the introduction, the BBB is a complex multi-

component structure that is likely to have several critical requirements for in vitro

culture to correctly model its functions. Enhancing the brain endothelial cell phenotype

of iPSC-derived endothelial cells and optimization of iPSC-derived BBB model is far

from complete. Improvement of models is an ongoing process. Likely, there are

opportunities for optimization of the differentiation and culture processes, which could

be exploited to further improve the BBB phenotype of the model and produce iPSC-

derived brain endothelial cells with more similar transcription signature to brain

endothelial cells in vivo. In addition, there is a great need to further characterize the

capacity of iPSC-derived BBB models to be used in permeability assessments. More

information about activity and expression is required for many of the transporter

proteins active at the BBB.

Brain permeability prediction in drug discovery

The current strategy for CNS permeability assessment in drug discovery relies on, first,

determining if the substance is an efflux transporter substrate and second, determining

the in vivo brain exposure in rodents. This is commonly preceded by in silico

modelling of BBB permeability used in the lead generation process. Efflux transporter

assays are generally performed using the low permeability human epithelial colorectal

adenocarcinoma cell line (Caco-2) and Madin-Darby canine kidney cells

overexpressing efflux transporter P-gp (MDCK-MRD1) line in Transwell systems

relatively early in the drug discovery process. Later, in vivo rodent permeability

assessments are performed. The ratio of the total brain concentration to total plasma

concentration at equilibrium combined with the fraction unbound in brain and fraction

unbound in plasma is determined. After infusion the concentrations in blood and in

whole brain homogenate are analysed, brain binding is typically assessed by incubating

rat brain slices with a compound cocktail. These methods are very low throughput and


59

require several animals per data point. Hence, they are performed at the last stages of

drug development before clinical trials.

iPSC-derived BBB models described in this thesis could replace the Caco-2 and

MDCK-MDR1 lines in efflux transporter assays. In contrast to Caco-2 and MDCK cell

lines, these iPSC-derived BBB models contain human brain-specific cells with

expression of many BBB transporters lacking in the Caco-2 and MDCK-MDR1 lines

which originate from colon and kidney respectively. Using human brain-specific cells

provides an opportunity to evaluate other transport routs in addition to efflux transport.

Additionally, using microfluidic iPSC-derived BBB models allows for higher

throughput analysis than a Caco-2 or MDCK-MDR1 Transwell assay. Consequently,

a microfluidic iPSC-derived BBB model have the possibility to provide important

information earlier in the drug discovery process. Earlier prediction of brain exposure

by a combination of mechanisms rather than efflux only would generate better

translatability to the rodent in vivo models, causing fewer undesired compounds to

make it as far as the in vivo assay. If fewer substances require in vivo model testing it

would both reduce the number of animals needed for testing and provide a more cost-

efficient process. Even though it would be desirable to replace in vivo animal testing

completely, that is not likely to transpire in the near future. Due to the inability of

present in vitro cell models to estimate brain-binding and metabolism, which govern

the unbound drug concentration. These models are not able to predict the amount of

substance which exerts the physiological function, i.e. the unbound fraction.

Furthermore, there are regulatory requirements for animal testing before human trials

and human data to verify an in vitro model to a satisfactory extent is lacking.

Consequently, CNS permeability assessment in drug discovery would benefit from the

use of an iPSC-derived BBB model in efflux assays. However, for the added benefit

of modelling additional transport processes a more extensive analysis of what transport

routes are accurately modelled in the iPSC-derived BBB model would need to be

performed beforehand. Such analysis should include gene and protein expression of

transporters together with functional analysis of transport compared to human in vivo

data. The major challenges would be finding validated substrates for all transporters

and the large amount of work it would take to generate the corresponding human in

vivo data. However, recent advances in integrated transcriptomic and proteomic

analysis and non-invasive brain PET imaging provides possible strategies to overcome

these challenges.

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Limitations

There are differences between iPSC lines depending on, for example, their genetic

background, what method was used for generation and culture conditions. In addition,

commercially available iPSC lines originate mostly from Caucasian male individuals.

This limits the ability to draw general conclusions. In this work, multiple iPSC lines

have been used in experiments to allow for more general conclusions. However, the

diversity of the genetic background in the lines used was still limited. There are further

limitations to the use of iPSC-derived cell types as they generally display an immature

or foetal-like phenotype. Hence, characterization of the function investigated needs to

be thorough. In this work complementary analysis of RNA, protein and function

increases confidence in cell type identity of iPSC-derived cell types. However,

analyses on all levels were not performed for all experiments, which constitutes a

limitation of what conclusions can be drawn. Specifically, the pathway analysis and

gene expression analysis in Paper I needs experimental verification to allow more

definite conclusions. Investigating differences in pathways and processes by

evaluation of annotated mRNAs can provide indication of changes because most of the

components of the pathway or process are evaluated, but experimental verification is

still needed.

Primary brain endothelial cells do not retain BBB phenotype in vitro, hence, they are

not suitable for comparison. Additionally, BBB permeability has historically required

invasive sampling and human data is very rare. The difficulties in comparing results

with both human primary cell culture and human in vivo data are major limitations of

this work. Many of the results from experiments in this work need further experimental

verification and confirmation via in vivo studies.

A large part of CNS permeability and availability assessment of drug substances are

measurements of unbound drug concentrations in the brain. The unbound drug

concentration is essentially the active concentration available to exert its physiological

effect. The permeability measurements performed in the in vitro models in this thesis

do not consider the binding and metabolism that may occur in the CNS affecting the

concentration of substance available in the CNS in vivo.

Models in this work were evaluated based on BBB-specific phenotype, however, no

challenges to the BBB were performed. Cocultures have been shown to increase the

tolerance of BBB models to challenges that could alter their permeabilities, but no such


61

affects were investigated in these studies. Consequently, coculture may have additional

effects only detectable under stress conditions not detected in these studies.

Delsing, Louise

62


63

9 CONCLUSIONS

In this thesis, iPSC-derived models of the BBB were developed and evaluated. Model

improvements were generated by evaluation of different methods to derive endothelial

cells and astrocytes from iPSC as well as evaluating different culture systems. A

microfluidic model was developed, which is compatible with efflux transporter assays

in high-throughput. The BBB models displayed a barrier phenotype in expression of

proteins and mRNA associated with brain endothelial cells. Functional testing showed

that the model exhibits selective permeability of both passively diffused substances

and substances that interact with brain-specific transporters. These properties were

comparable to other iPSC-derived models and in vivo permeability restriction. The

outcomes provide new insight into molecular mechanisms that influence iPSC-derived

BBB models’ ability to restrict permeability and to model requirements for

permeability assessments. The models developed in this thesis provide a promising

starting point for the use of iPSC-derived BBB models in drug discovery and

permeability assessments. In addition, the work highlights remaining questions and

challenges for iPSC-derived BBB models that need to be addressed for the models to

be useful in a wider range of applications. In particular, there is a need for standardizing

permeability assessment assays across models and to increase the number of

comparisons to in vivo human data.

Delsing, Louise

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65

10 FUTURE PERSPECTIVES

Building on the work in this thesis there are several opportunities to further

characterize and improve iPSC-derived BBB models. In Paper IV, we showed that the

microfluidic model has some improved barrier restriction properties and can be

implemented in high-throughput assays. However, many questions remain about what

changes can be observed in the microphysiological model compared to the static

Transwell culture. A deeper investigation of how the culture environment and the shear

stress in the microphysiological model affect the cells would be highly interesting. A

genome wide expression profiling of mRNA and protein comparing the two models

would allow for an analysis of what BBB properties are affected by MPS culture and

could help direct further mechanistic studies. Additionally, establishing a coculture

model in the MPS system may further improve the BBB model in a similar manner

seen in many Transwell static models. Indeed, reduced permeability was observed in

a recent iPSC-derived BBB MPS model after coculture with astrocytes, neurons and

neural progenitors (133). By applying pericytes and astrocytes in the gel compartment

of the MPS system, established in Paper IV, a contact coculture, similar to that in vivo

could be formed. An isogenic BBB model could be created using the recently

published protocol for derivation of brain-specific pericytes (99) and using the xeno-

free derivation of astrocytes described in Paper III. The collagen gel used for gel

casting in the MPS and the brain pericyte protocol contain some animal components

and needs to be modified to xeno-free conditions before a fully defined, isogenic iPSC

BBB model can be derived. Recently, a serum free protocol for differentiation to brain

endothelial cells was published which could be adopted in future BBB models (91). A

xeno-free BBB model would likely reduce variability and be an important optimization

step for the coculture model to be used in high-throughput permeability assays.

The number of iPSC-derived models of the BBB is rapidly increasing, but the use of

iPSC-derived BBB models in drug discovery is still limited. While using iPSC-derived

cells allow for studying models with a diseases background and comparing to

genetically modified isogenic controls it is still likely that the first large-scale use of

these models will be permeability assessment. Recreating a disease phenotype, in vitro,

in a complex multicellular system such as the BBB is still a great challenge. Major

issues include recreating the specific structure of the BBB that is needed for its

function, optimizing culture conditions to several cell types, variability in cell culture

and differentiation, and providing a biologically relevant model in a usable screening

Delsing, Louise

66

format. Creating a model for permeability assessment may still require complex in

vitro cultures, but quality control standardization could more easily be adopted for one

functional readout than a complex multifaceted disease phenotype. Standardizing the

characterization and validation of models would enhance their application and

adoption within drug discovery. Such standardization may include establishing

analytical performance standards for models and a defined set of reference compounds

that can demonstrate desired outcome and be compared across different models and

labs. The lack of human in vivo permeability data for comparison is a major hurdle in

model development. To establish a reference set of compounds for permeability

testing, human in vivo data needs to be obtained so that comparisons can be made. In

recent years, quantitative imaging permeability assessments in humans have increased

and provide a non-invasive way of measuring brain penetration of substances. The

increasing amount of human in vivo data generated with this method may facilitate

human in vivo to in vitro comparisons in the future. After all, for these models to be

successfully adopted by the pharmaceutical industry they need to have good predictive

capacity, sufficient throughput and compatibility with automated handling, low

variability and ease of use.

There are many potential future applications of iPSC-derived BBB models, especially

in modelling the complex cellular cross talk between different cell types at the BBB.

There have been substantial investments in research on how neuronal cells and

pericytes influence the BBB formation, function, and maintenance. However recent

literature suggests that endothelial cells at the BBB may play a significant role in the

communication between the peripheral organs and the CNS, both via the proteins

secreted by the endothelial cells into the CNS and regulation of the controlled transport

across the BBB. It has been shown, in an iPSC-derived system, that the vasculature

has specific maturation effects on spinal motor neurons (151), and in the adult central

nervous system the vasculature regulates neural stem cell behaviour by providing

circulating and secreted factors. Age-related decline of neurogenesis and cognitive

function is associated with reduced blood flow and decreased numbers of neural stem

cells. Therefore, restoring the functionality of the CNS vasculature could counteract

some of the negative effects of aging. It has been shown that factors found in young

blood induce vascular remodelling, culminating in increased neurogenesis and

improved olfactory discrimination in aging mice. Remarkably, one of the identified

substances contributing to these effects does so without entering the CNS itself (152).

Remodelling of the brain vasculature may function as a mediator in providing benefits


67

such as increased neurogenesis and improved cognition and hence, brain endothelial

cell secreted proteins may be of high importance. iPSC-derived BBB coculture models

could be a useful tool to further explore how signalling from the brain endothelium

affects neurons and other CNS components.

Another highly interesting feature of the BBB, not examined in this work, is how the

nutrient supply to the brain across the BBB is affected in aging and neurodegenerative

disorders. The brain accounts for 20% of all energy consumption at rest. Glut-1 is

responsible for a majority of the glucose uptake from the blood to the brain and brain

glucose uptake correlates with Glut-1 levels (71, 153). As shown in Papers I, III and

IV the high Glut-1 level in the BBB are recapitulated in the iPSC-derived model and

thus it may be a good candidate model for studies of the Glut-1 mediated transport.

Indeed, active glucose uptake through Glut-1 has been shown in iPSC-derived brain

endothelial cells derived with similar methods (154). Ideally, iPSC-derived cells from

a disease background and their isogenic controls could be used for such studies.

Mutations in the Glut-1 gene SLC1A2, also known as Glut-1 deficiency syndrome

cause seizures, delayed development and microencephaly, due to low CSF glucose

levels (155). Reduction in Glut-1 levels and glucose transport have been observed in

animal models of both aging and AD (156, 157) and in AD patients (158).

Furthermore, glucose uptake is reduced in individuals with genetic risk for AD (159).

Glucose metabolism is reduced in individuals with a family history of AD (160) and

cognitively normal individuals who later develop AD (161). Consequently, reduced

glucose transport has been suggested to precede AD onset and affect the progression,

BBB stability and pathology in AD (162). Increasing the understanding of glucose

transport deficits in healthy and diseased individuals could be useful both in terms of

earlier diagnosis and exploration of new therapeutic strategies.

In conclusion, the iPSC-derived BBB model systems are still in their early

development, this is especially true for MPS. These systems have great capacity to

advance into highly sophisticated models and there will indubitable be many new

applications for these systems in the future. However, many challenges still remain,

particularly with respect to reproducibility and recreation of multifaceted phenotypes

in vitro with increasing complexity in the models. An important first step towards

improved BBB models would be to establish analytical performance standards that can

be compared with in vivo human data and across model systems.

Delsing, Louise

68


69

ACKNOWLEDGEMENT

This work would not have been possible without the help and encouragement from a

large group of colleagues, friends and family. I would like to express my sincerest

gratitude to all of you.

First to my supervisors,

Jane, thank you for always asking questions and for your constant curiosity. The way

you lead projects and produce consensus is incredible.

Ryan, thank you for straightening out my thoughts and plans when they become to

intertwined and messy. Your leadership and coaching skills are a true inspiration.

Henrik, thank you for your endless enthusiasm and creative ideas.

Gabriella, thank you for believing in me and always promoting me.

To the past and present members of the Stem and Primary Cell Team at AstraZeneca,

you make coming to work every day a joyful experience. The warm and welcoming

atmosphere that all of you create is unique and I am lucky to have spent my 4 years as

a PhD student in your company. Cecilia, thank you for being a mentor in the true sense

of the word, always brining good advice and encouragement. Sharing an office with

you for most of these four years has been an absolute pleasure. Anna S, thank you for

always being kind and offering to help. Anette, thank you for being a great friend and

bringing joy wherever you go. Shailesh, thank you for advice in neuroscience and for

spreading happiness in the corridor. Anna F, thank you for making sure our lab runs

smoothly, your work is invaluable. Anna J, thank you for caring and coaching, thank

you for bringing enthusiasm in the lab. Louise S, thank you for taking the time to help

me out whenever I had trouble with my cell cultures, what you do not know about cell

culture is not worth knowing. Linnea and Sebastian, thank you for bringing enthusiasm

for MPS to the team. Farideh, thank you for taking an interest in my work and always

asking questions. Yasaman, thank you for your kindness and caring personality. Linn,

thank you for all the cell culture help with the final manuscript. Anders, thank you for

so many valuable scientific discussions and for being an inspiration.

Thank you, Maryam and José, for help with RNAseq, Marie and Mattias for help with

mass spectrometry, Alan for help with calcium signalling analysis and Mei for help

with inflammatory response analysis.

To Anna Herland at KTH, thank you for taking an interest in my project and for taking

the time to give great advice. Talking to you for 15 minutes has been enough to figure

Delsing, Louise

70

out issues I struggled with for weeks. I cannot thank you enough. To Dimitris at KTH,

thank you for your valuable input on manuscripts. To Anna Falk at Karolinska, thank

you for your great advice and for generously allowing me to use your cell lines.

To the past and present members of the TransBIG team at Skövde University. Thank

you to Markus, Nidal, Gustav, Jonas, Claudia, Peter and Sepideh for your support,

enthusiasm and advice. Thank you for the fun times at ISSCR. Thank you to Benjamin

for explaining the ins and outs of RNAseq data processing. Thank you, Pierre, for

assistance with RNAseq data analysis. Thank you to Björn, Angelika, Selmina,

Tejaswi, Dirk and Julia for nice trips to Kvänum and for taking an interest in my work.

To the collaborators in the BBB project, Therese at BioLamina and Carl and Jan at

3Dtro, thank you for advice and input.

To my family, thank you to my mother Désirée and my father Per for your endless

support and encouragement. Thank you to my sisters, Mimmi, Christine and Anna for

cheering me on, in happy moments and when I was struggling.

Last but not least, to my husband Daniel. Thank you for reminding me about what is

important in life. Thank you for your patience and understanding during weekends I

spent in the lab. Thank you for always being there, regardless if that means helping me

deal with failure or celebrate success. I could not have done it without you.


71

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